IMMUNE RESPONSES TO HUMAN NOROVIRUS AND HUMAN NOROVIRUS
VIRUS-LIKE PARTICLES IN GNOTOBIOTIC PIGS AND CALVES
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the
Degree Doctor of Philosophy in the Graduate School of
The Ohio State University
By
Menira B. L. Dias e Souza, M.S.
*****
The Ohio State University 2007
Dissertation Committee:
Distinguished University Professor Dr. Linda J. Saif, Adviser
Associate Professor Dr. John H. Hughes Approved by
Adjunct Assistant Professor Dr. Lijuan Yuan ______
Adviser Graduate Program in Veterinary Preventive Medicine
ABSTRACT
The Caliciviridae family is constituted of four distinct genera: Norovirus,
Sapovirus, Lagovirus, and Vesivirus. The Noroviruses (NoVs) are classified within 5
genogroups (GI-V) and at least 27 genotypes, based on the partial capsid, and regions
A-D of the RNA dependent RNA polymerase. Caliciviruses (CV) infect various hosts and cause a wide spectrum of diseases. The human noroviruses (HuNoV) are transmitted by the fecal-oral route and constitute the leading cause of epidemic food and water-borne non-bacterial gastroenteritis worldwide. They are generally highly stable in the environment, which contributes to their dissemination and consequently to disease
impact.
The HuNoV disease is characterized by nausea, vomiting and abdominal cramps.
These symptoms are usually self-limiting and cease within 24-48 hrs. However, these
agents are responsible for great disease burden in both developed and developing
countries, affecting people of all ages.
The determinants of susceptibility and/or resistance to HuNoV are not completely
understood; however recently, the histo-blood group type and secretor status were
identified as genetic factors associated with risk of Norwalk-virus infection and disease.
Numerous HuNoV outbreaks have been detected in the past decade and the most
ii common sources are contaminated food and water, with secondary person-to-person
transmission also being a common cause. These outbreaks take place in various settings
such as: day-care centers, hospitals, cruise ships and schools. Outbreaks are also common
among certain populations that live in close contact and in isolation such as the military personnel and elderly persons in nursing homes, causing high morbidity and low mortality. Therefore, the use of an efficacious and safe vaccine against HuNoV, especially among these populations would be of great benefit. The SaV and NoV have great genetic diversity and are fastidious viruses that do not grow in routine cell culture, except for the murine norovirus (MNV), and the porcine enteric calicivirus (PECV).
Their extensive genetic diversity and fastidious character contribute to the diagnose confusion and limited knowledge about these viruses replication strategies, pathogenesis and host immune responses. However; recently, important progresses has been made with the in vitro growth of GI and GII HuNoV strains, using a 3-dimensional organoid model of human intestinal epithelium, and the successful replication of the HuNoV GII.4-HS66 strain in the gnotobiotic (Gn) pig model. Therefore, the use of experimental animal models constitutes an alternative for the study of HuNoV and is an important tool for a better understanding of HuNoV replication, pathogenesis, virus-host interactions and host immune responses to these agents.
Our first objective was to evaluate the antibody and cytokine immune responses, both locally (intestine) and systemically to the GII.4 HuNoV-HS66 strain using the gnotobiotic (Gn) pig model. Low antibody titers and antibody-secreting cell (ASC) numbers were elicited in the HuNoV-HS66-inculated pigs, but 65% of the pigs seroconverted. A higher Th1 (high IL-12 but low and transient IFN-γ), but also low Th2
iii (IL-4 and IL-10), low transient pro-inflammatory (IL-6) cytokine and delayed innate
(IFN-α) responses were induced by the HuNoV-HS66 in the serum of the Gn pigs.
Intestinal IFN-α and IL-12 (late) were also significantly elevated after infection. Higher
numbers of Th1 (IL-12 and IFN-γ) cytokine-secreting cells (CSC) were elicited when
compared to Th2 (IL-4) and pro-inflammatory (IL-6) cytokines.
Our second objective was to evaluate the potential of the Gn calf as an alternative
model to study the pathogenesis and host immune responses after oral inoculation with
the HuNoV-HS66 strain. Isotype-specific (IgM, IgA and IgG) antibody levels and the
concentrations of the pro-inflammatory (TNF-α), Th1 (IL-12 and IFN-γ) and Th2 (IL-4) and Th2/T-reg (IL-10) cytokines were determined, at selected post-inoculation days
(PIDs) in the serum, fecal samples and IC of the Gn calves, together with the CSC numbers in the intestine, mesenteric lymph nodes, spleen and blood at PID 28. The
HuNoV-HS66 caused diarrhea and intestinal lesions, mainly in the proximal part of the intestine of one of the calves euthanized at PID 3. Viral shedding was detected from post- inoculation day 1-6 and 67% of the animals seroconverted with HuNoV-HS66-specific
IgA and IgG antibodies. Both Th1 (IL-12 and IFN-γ) and Th2/T-reg (IL-10) cytokines, but also pro-inflammatory (TNF-α) were induced in the serum and fecal samples of the
HuNoV-HS66-inoculated calves.
Our third objective was to evaluate the antibody and cytokine immune responses in Gn pigs, both locally and systemically, to a HuNoV GII.4 (HS66 strain) virus-like particle (VLP) vaccine using 1 oral and 2 IN doses (250 μg/dose), in conjunction with the
mucosal adjuvants, ISCOM or mLT, compared to controls (each adjuvant alone). We also
evaluated the protection induced by both vaccination regimens to homologous viral
iv challenge in pigs of A+/H+ phenotype. A 100% seroconversion rate was observed in the vaccinated pigs, regardless of the vaccine regimen, and 100% of coproconversion was detected in the VLP+ISCOM pigs compared to 75% of the VLP+mLT group. However, only 57% of the control pigs shed virus post-challenge. Pre-challenge, the VLP+ISCOM vaccine induced higher IgA and IgG ASC both systemically and locally and IgA and IgG in IC, whereas the VLP+mLT induced higher systemic Th1 and Th2 CSC numbers and highest Th1 (IFN-γ) cytokine responses in IC. Thus, the VLP+mLT vaccine induced a balanced Th1/Th2 response, whereas the VLP+ISCOM induced a more Th2 biased response, but both vaccine regimens induced high levels of protection against virus shedding and diarrhea
In summary, this was the first study to delineate in detail both local and systemic immune responses to a GII.4 HuNoV (HS66 strain) using the Gn pig and calf model and to describe the pathogenesis of this strain in Gn calves. This was also the first study to evaluate the immunogenicity and protective potential of GII HuNoV VLP vaccines and also the protection induced by these vaccines after viral homologous challenge of the Gn pigs.
v
Dedicated to my husband Murillo for making me a better person and for embracing my dreams as if they were his own
To my family and friends
for their unconditional love and support through this journey
vi ACKNOWLEDGMENTS
I thank my adviser, Dr. Linda J. Saif for her guidance; support and constructive criticism
that helped me grow intellectually through this process
Thanks to my committee members Dr. John H. Hughes and Dr. Lijuan Yuan for their
helpful suggestions and contributions to this work
I also would like to thank Dr. Divina Cardoso for introducing me to the scientific world
I am greatly thankful to Dr. Marli Azevedo, Dr. Ana Gonzales and Veronica Costantini
for their friendship and for sharing their knowledge
Special thanks to Dr. Sonia M. Cheetham and Kwonil Jung for their contributions to this
work
I also thank Dr. Juliet Hanson, Peggy Lewis, Richard McCormick, Janet McCormick,
Greg Meyers and Todd Root for technical support and for their dedication to the Food
Animal Health Research Program
Thanks to all my former and present lab colleagues Dr. Anastasia Vlasova, Dr. Kostanini
Alekseev, Dr. Jason Zhang, Dr. Li Guohua, Wei Zhang, Dr. Myung Guk Han and Dr.
Qiuhong Wang for all their help, understanding and companionship
vii VITA
October 29th, 1973 Born - Brasilia, DF, Brasil
1991-1996 B.S in Biological Sciences, Catholic University of Goias Goiania, Goias, Brasil
1996-1998 Lab. Technician at PADRAO Clinical Laboratory Goiania, Goias, Brasil
1999-2001 M.S Tropical Medicine, Federal University of Goias Goiania, Goias, Brasil
2002- present Graduate Research Associate Food Animal Health Research Program Ohio Agricultural Research and Development Center Department of Veterinary Preventive Medicine The Ohio State University Wooster, Ohio
PUBLICATIONS
1. Cheetham, S., Souza, M., Meulia, T., Grimes, S., Han, M.G. and Saif L. J. (2007) Binding patterns of human norovirus-like particles to buccal and intestinal tissues of gnotobiotic pigs in relation to A/H histo-blood group antigen expression. J Virol 81, 3535-3544.
2. Cheetham, S., Souza, M., Meulia, T., Grimes, S., Han, M.G. and Saif L. J. (2006) Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs. J Virol 80, 10372- 10381. 3. Wang, Q., M., Souza, J.A. Funk., W. Zhang, and L.J. Saif. (2006) Prevalence of noroviruses and sapoviruses in swine of various ages determined by reverse transcription- PCR and microwell hybridization assays. J Clin Microbiol 44, 2057-2062.
4. Wang, Q., M.G. Han, S. Cheetham, M. Souza, J.A. Funk, and L.J.Saif. (2005). Porcine noroviruses related to human noroviruses. Emerg Infect Dis 11, 1874-1881.
viii 5. Costa, P.S.S., Cardoso, D.D.C., Grisi, S.F.F.E., Silva, P.A., Fiaccadori, F.S., Souza, M.,B.L.D., and Santos, R. A. T. (2004). Manifestacoes clinicas e epidemiologicas das infeccoes por Rotavirus A. Pediatria (USP), Brasil, 26, 151-158.
6. Costa, P.S.S., Cardoso, D.D.P., Grisi, S.J.F.E., Silva, P.A., Fiaccadori, F.S., Souza, M.B. L., and Santos, R.A.T. (2004) Infeccoes e reinfeccoes por Rotavirus A: genotipagem e implicacoes vacinais. Jornal de Pediatria, Brasil 80, 119-122.
7. Souza, M.B.L.D., M. L. Racz, J. P. G. Leite, C. M. A. Soares, R. M. B. Martins, V. Munford, and D. D. P. Cardoso. (2003). Molecular and Serological Characterization of Group A Rotavirus Isolates Obtained from Hospitalized Children in Goiania, Brazil, 1998-2000. Eur J Clin Microbiol Infect. Dis 22, 441-43.
8. Cardoso, D. D.P., F. Fiaccadori, M. B. L. D. Souza, R. M. B. Martins and J. P. G. Leite. 2002. Detection and genotyping of astroviruses from children with acute gastroenteritis from Goiania, Goias, Brazil. Med. Sci. Monit 8, CR624-8.
9. Cardoso, D. D., C. M. Soares, M. B. D. e Souza, M. da S. De Azevedo, R. M. Martins, D. A. Queiroz, W. M. de Brito, V. Munford and M. L. Racz. 2003. Epidemiological features of rotavirus infection in Goiania, Goias, Brazil from 1986 to 2000. Mem. Inst. Oswaldo Cruz 98, 25-9.
FIELDS OF STUDY
Major Field: Veterinary Preventive Medicine
Studies in Immunology and Virology
ix
TABLE OF CONTENTS Page Abstract……………………………………………………………………………………ii
Dedication………………………………………………………………………………...vi
Acknowledgments……………………………………………………………………….vii
Vita……………………………………………………………………………………...viii
List of tables……………………………………………………………………………..xix
List of figures…………………………………………………………………………….xx
List of abbreviations……………………………………………………………………xxii
Chapters:
CHAPTER 1: LITERATURE REVIEW ON ENTERIC CALICIVIRUSES AND
IMMUNITY
1.1 Introduction and History………………………………………………………………1 1.1.1 Norwalk virus discovery…………………………………………………….1 1.1.2 Early volunteer studies………………………………………………………2 1.1.3 Recent volunteer studies…………………………………………………….2
1.2 Classification…………………………………………………………………………..3 1.2.1 Taxonomy and genome organization………………………………………..3
1.3 Viral structure…………………………………………………………………………6 1.3.1 Structural proteins…………………………………………………………...6 1.3.2 Nonstructural proteins……………………………………………………….8
x 1.3.3 Virus-like particles (VLPs)………………………………………………...11 1.4 General biological features…………………………………………………………..12 1.4.1 Viral stability and infectivity………………………………………………13 1.4.2 In vitro viral replication……………………………………………………14 1.4.3 Calicivirus infectious clones……………………………………………….16
1.5 Diagnosis……………………………………………………………………………..18
1.6 Animal caliciviruses………………………………………………………………….23 1.6.1 Porcine sapovirus…………………………………………………………..23 1.6.2 Porcine norovirus…………………………………………………………..25 1.6.3 Bovine norovirus and other enteric caliciviruses: NB strain………………26 1.6.4 Murine norovirus…………………………………………………………..28
1.7 Pathogenesis………………………………………………………………………….30 1.7.1 Clinical features……………………………………………………………30 1.7.2 Incubation period and viral shedding………………………………………31 1.7.3 Viral replication……………………………………………………………31 1.7.4 Susceptibility and resistance to HuNoV infection…………………………36 1.7.5 Virulence and persistence………………………………………………….40 1.7.6 Apoptosis…………………………………………………………………..42
1.8 Epidemiology………………………………………………………………………...44 1.8.1 Molecular epidemiology…………………………………………………...44 1.8.2 Seroprevalence……………………………………………………………..46 1.8.3 Incidence…………………………………………………………………...49 1.8.4 Viral outbreaks……………………………………………………………..51 1.8.4.1 Sources of NoV outbreaks……………………………………….53 1.8.5 Interspecies transmission and zoonosis……………………………………57
xi 1.9 Immunity……………………………………………………………………………..58 1.9.1 T cells………………………………………………………………………58 1.9.2 B cells……………………………………………………………………....61 1.9.3 Dendritic cells……………………………………………………………...63 1.9.4 Cytokines…………………………………………………………………..65
1.10 Mucosal immunity………………………………………………………………….70 1.10.1 Mucosal immune system………………………………………………….70 1.10.1.1 NALT…………………………………………………………...71 1.10.1.2 GALT…………………………………………………………...73 1.10.1.3 IgA……………………………………………………………...80 1.10.1.4 T and B cell homing…………………………………………….81
1.11 Mucosal vaccines…………………………………………………………………...83 1.11.1 Adjuvants and antigen delivery systems for mucosal vaccines…………..84 1.11.1.1 Mucosal adjuvants……………………………………………...84 1.11.2 Mucosal antigen delivery systems………………………………………..89
1.12 Immunity to caliciviruses…………………………………………………………...91
1.13 Treatment and prevention…………………………………………………………..95 1.13.1 Treatment…………………………………………………………………95 1.13.2 Preventive measures………………………………………………………98
1.14 Vaccines…………………………………………………………………………….99 1.14.1 Animal studies……………………………………………………………99 1.14.2 Human volunteer studies………………………………………………...104
1.15 References…………………………………………………………………………108
xii CHAPTER 2: CYTOKINE AND ANTIBODY RESPONSES IN GNOTOBIOTIC PIGS AFTER INFECTION WITH HUMAN NOROVIRUS GENOGROUP II.4- HS66 STRAIN
2.1 Summary……………………………………………………………………………153
2.2 Introduction…………………………………………………………………………154 2.3 Materials and Methods……………………………………………………………...156 2.3.1 Virus inoculum……………………………………………………………156 2.3.2 Inoculation of the experimental pigs……………………………………...157 2.3.4 Assessment of diarrhea…………………………………………………...158 2.3.5 Detection of viral shedding by RT-PCR………………………………….158 2.3.6 Detection of viral shedding by antigen-ELISA…………………………..159 2.3.7 Viremia…………………………………………………………………...159 2.3.8 Antibody detection……………………………………………………….159 2.3.9 Isolation of MNC for antibody and cytokine-secreting cells ELISPOT assays……………………………………………………160 2.3.10 ELISPOT assay for HuNoV-HS66-specific antibody-secreting cells (ASC)……………………………………….…160 2.3.11 Cytokine-secreting cells (CSC) ELISPOT assay………………………..161 2.3.12 Cytokine ELISA assay…………………………………………………..161 2.3.13 Statistical analysis……………………………………………………….162
2.4 Results………………………………………………………………………………163 2.4.1 HuNoV-HS66 induces diarrhea, rectal shedding and viremia in Gn pigs……………………………………………………163 2.4.2 Inact-HS66 does not induce diarrhea, rectal shedding or viremia in Gn pigs……………………………………………………..163 2.4.3 HuNoV-HS66 elicits low antibody responses in Gn pigs………………..164 2.4.4 Seroconversion, serum and intestinal convalescent antibody
xiii titers to HuNoV-HS66 are associated with diarrhea severity during the acute phase of infection………………………………………165 2.4.5 HuNoV-HS66 induces low numbers of HS66-specific ASC systemically and locally in Gn pigs………………………………...165 2.4.6 Local ASC responses were strongly correlated with systemic ASC responses after HuNoV-HS66 infection………………….166 2.4.7 HuNoV-HS66 induced a balanced Th1/Th2 and a delayed Type I IFN response in serum of Gn pigs………………………………...166 2.4.8 Only type I IFN (early and late) and IL-12 (late) cytokine concentrations were significantly elevated in the IC of the HuNoV-HS66 pigs when compared to controls….………………..167 2.4.9 HuNoV-HS66 elicited higher numbers of Th1 (IL-12 and IFN-γ) CSC when compared to Th2 (IL-4) and pro-inflammatory (IL-6) CSC both systemically and locally………..168 2.4.10 Systemic HuNoV-HS66 responses strongly correlated with local HuNoV- HS66 CSC responses………………………………..169 2.5 Discussion…………………………………………………………………………..170
2.6 Acknowledgements…………………………………………………………………178
2.7 References…………………………………………………………………………..179
CHAPTER 3: PATHOGENESIS, AND IMMUNE RESPONSES IN GNOTOBIOTIC CALVES AFTER INFECTION WITH HUMAN NOROVIRUS GENOGROUP II.4-HS66 STRAIN
3.1 Summary……………………………………………………………………………191 3.2 Introduction…………………………………………………………………………192 3.3 Materials and Methods……………………………………………………………...195 3.3.1 Virus inoculum……………………………………………………………195
xiv 3.3.2 Inoculation of the experimental calves………………………………...... 196 3.3.3 Assessment of diarrhea…………………………………………………...196 3.3.4 Histopathology……………………………………………………………197 3.3.5 Viral antigen detection by immunohistochemistry……………………….197 3.3.6 Detection of viral shedding by RT-PCR………………………………….198 3.3.7 Detection of viral shedding by antigen ELISA…………………………...199 3.3.8 Viremia…………………………………………………………………...199 3.3.9 Antibody detection……………………………………………………….199 3.3.10 Isolation of MNC for ELISPOT assays to detect antibody and cytokine secreting cells…………………………………………………200 3.3.11 ELISPOT assay for HuNoV-specific antibody-secreting cells (ASC)….200 3.3.12 Cytokine-secreting cell (CSC) ELISPOT assay………………………...201 3.3.13 Cytokine ELISA assay…………………………………………………..202 3.3.14 Statistical analysis……………………………………………………….203
3.4 Results………………………………………………………………………………203 3.4.1 The HuNoV-HS66 caused intestinal lesions in the jejunum of a Gn calf……………………………………………...…………………..203 3.4.2 Viral antigen was detected by immunohistochemistry (IHC) (IHC) in the jejunum of a HuNoV-HS66-inoculated calf…….………….204 3.4.3 HuNoV-HS66 caused diarrhea, viral shedding and viremia in Gn calves……………………………………...………………………..204 3.4.4 HuNoV elicited 67% seroconversion and 33% coproconversion rates in Gn calves……………………………………………………………….205 3.4.5 HuNoV-HS66 induced higher numbers of IgA and IgG ASC locally compared to systemically……………….……….………….206 3.4.6 HuNoV-HS66 induced a significant early peak (PID2) of IFN-γ in serum of Gn calves…………………..………………………………...206 3.4.7 HuNoV-HS66 induced in Gn calves high numbers of IFN-γ CSC Both locally (MLN) and systemically (spleen), high numbers
xv of pro-inflammatory (TNF-α) systemically (spleen and blood) and high numbers of Th2/T-reg (IL-10) both locally (intestine) and systemically (spleen)………………………..…………….207 3.4.8 HS66-specific CSC responses detected by cytokine ELISPOT………….208
3.5 Discussion…………………………………………………………………………..209
3.6 Acknowledgments…………………………………………………………………..218
3.7 References…………………………………………………………………………..219
CHAPTER 4: A HUMAN NOROVIRUS-LIKE PARTICLE VACCINE ADJUVANTED WITH ISCOM OR MLT INDUCES CYTOKINE AND ANTIBODY RESPONSES AND PROTECTION TO HOMOLOGUS VIRUS IN A GNOTOBIOTIC PIG DISEASE MODEL
4.1 Summary……………………………………………………………………………231
4.2 Introduction…………………………………………………………………………232
4.3 Materials and Methods……………………………………………………………...235
4.3.1 Recombinant HS66 VLPs………………………………………………...235 4.3.2 VLP-ISCOM vaccine……………………………………………………..236 4.3.3 VLP-mLT vaccine………………………………………………………..237 4.3.4 HuNoV-HS66 virus challenge inoculum…………………………………237 4.3.5 Inoculation of the experimental pigs……………………………………...237 4.3.6 A/H typing………………………………………………………………..238 4.3.7 Assessment of diarrhea…………………………………………………...238 4.3.8 Viral shedding by RT-PCR……………………………………………….239
xvi 4.3.9 Viral shedding by antigen-ELISA………………………………………..239 4.3.10 Antibody detection by immunocytochemistry…………………………..239 4.3.11 Isolation of mononuclear cells (MNC) for ELISPOT assays…………...240 4.3.12 ELISPOT assay for detection of HuNoV-HS66-specific antibody-secreting cells (ASC)…………………………………………240 4.3.13 ELISPOT assay for detection of HuNoV-HS66-specific cytokine-secreting cells (CSC)…………………………………………241
4.3.14 Cytokine concentration in the serum and IC quantitated by cytokine ELISA………………………………………………….……..242 4.3.15 Statistical analysis……………………………………………………….243
4.4 Results………………………………………………………………………………243 4.4.1 A/H phenotype of Gn pigs………………………………………………..243 4.4.2 The HuNoV-HS66 VLP vaccine protected pigs after HuNoV-HS66 challenge with the VLP+mLT vaccine inducing complete protection…...243 4.4.3 The HuNoV-HS66 VLPs induced systemic and intestinal antibody responses in Gn pigs regardless of the adjuvant used…………………….244 4.4.4 The HuNoV-HS66 VLPs elicited higher numbers of ASC responses locally (intestine) compared to systemically (spleen and blood) in the VLP+ISCOM vaccinated pigs…………………………246 4.4.5 Both VLP vaccines induced a significant increase in serum Th1 (IFN-γ) concentration only after viral challenge…………………………248 4.4.6 The Th1 cytokine (IFN-γ) was significantly elevated post-challenge in IC only in the VLP+mLT vaccinated pigs…………….249 4.4.7 Th1 (IL-12 and IFN-γ), Th2 (IL-4), Th2/T-reg (IL-10) and pro-inflammatory (IL-6) CSC were elicited at higher numbers systemically only in the VLP+mLT vaccinated Gn pigs pre-challenge….250
4.5 Discussion…………………………………………………………………………..251
xvii
3.6 Acknowledgments…………………………………………………………………..258
3.7 References………………………………………………………………………….260
CHAPTER 5: GENERAL SUMMARY AND CONCLUSIONS…………………..272
BIBLIOGRAPHY……………………………………………………………………..281
xviii LIST OF TABLES
Table Page 2.1 Diarrhea, fecal virus shedding and viremia detected by RT-PCR and serovonversion detected by immunocytochemistry in Gn pigs inoculated with either HuNoV-HS66 or mock-inoculated controls……………184
2.2 Cytokine responses (fold increase over controls) in serum of HuNoV-HS66-infected Gn pigs………………………………………………...185
3.1 Diarrhea, fecal virus shedding and viremia by RT-PCR and Seroconversion detected by immunocytochemistry in Gn calves Inoculated with either HuNoV-HS66 or mock-inoculated controls……………224
4.1.1 Fecal virus shedding, diarrhea, seroconversion, protection rates and coproconversion in Gn pigs vaccinated with either HuNoV-HS66 VLP+ISCOM, HuNoV-HS66 VLP+mLT or controls (ISCOM and mLT)…….265
5.1 Comparison between antibody titers in serum and intestinal contents and antibody-secreting cell numbers locally and systemically in HuNoV-HS66 orally inoculated pigs and in pigs vaccinated with 1 oral and 2 IN doses of either vaccination regimen (VLP+ISCOM or VLP+mLT)………………...………………………279
5.2 Comparison between cytokine concentrations in serum and intestinal contents and cytokine-secreting cell numbers locally and systemically in HuNoV-HS66 orally inoculated pigs and in pigs vaccinated with 1 oral and 2 IN doses of either vaccination regimen (VLP+ISCOM or VLP+mLT…..280
xix
LIST OF FIGURES Figure Page
2.1 Mean numbers of isotype-specific (IgM, IgA and IgG) antibody-secreting cell responsesin intestine, spleen and PBMC of gnotobiotic pigs inoculated with HS66 or controls………………………….186
2.2 Cytokine concentration in serum of gnotobiotic pigs inoculated with HuNoV-HS66 or controls…………………………………………………187
2.3 Cytokine concentration in intestinal contents of gnotobiotic pigs inoculated with HuNoV-HS66 or controls……………………………………..188
2.4 Pro-inflammatory (IL-6) and Th1 (IFN-γ and IL-12) cytokine secreting cell responses in intestine, spleen and PBMC of gnotobiotic pigs inoculated with HS66 or controls………………………….189
2.5 Th2 (IL-4) and Th2/T-regulatory (IL-10) cytokine secreting cell mean numbers in intestine, spleen and PBMC of gnotobiotic pigs inoculated with HS66 or controls………………………………………………190
3.1 Histological examination of the intestine of a mock-inoculated calf…………..225
3.2 Histological examination of the intestine of a HuNoV-HS66 inoculated calf….2254
3.3 Immunohistochemistry for detection of GII HuNoV capsid antigens………….226
3.4 Isotype-specific (IgM, IgA and IgG) antibody titers and and antibody-secreting cell responses in intestine, spleen and PBMC of gnotobiotic calves inoculated with HS66 or controls………………………..227
3.5 Th1 (IL-12 and IFN-γ), pro-inflammatory (TNF-α) and Th2/T-reg (IL-4 and IL-10) cytokine concentrations in serum of Gn calves inoculated with HuNoV-HS66 or controls………………………….228
3.6 Th1 (IL-12 and IFN-γ), pro-inflammatory (TNF-α) and Th2/T-reg cytokine concentrations in fecal samples of gnotobiotic calves inoculated with HuNoV-HS66 or controls……………………………………..229
xx 3.7 Th1 (IL-12 and IFN-γ), pro-inflammatory (TNF-α) and Th2/T-reg (IL-4 and IL-10) cytokine-secreting cell responses in intestine, MLN, spleen and blood of gnotobiotic calves inoculated with HuNoV-HS66 or controls……………………………………..230
4.1 Isotype-specific (IgM, IgA and IgG) antibody responses in the serum of gnotobiotic pigs vaccinated with 1 oral and 2 IN doses of each vaccination regimen (HuNoV-HS66VLP+ISCOM or mLT) or controls………………………………………………………………………266
4.2 Isotype-specific (IgM, IgA and IgG) antibody responses in the IC of gnotobiotic pigs vaccinated with 1 oral and 2 IN doses of each vaccination regimen (HuNoV-HS66VLP+ISCOM or mLT) or controls………………………………………………………………………266
4.3 Mean numbers of isotype-specific (IgM, IgA and IgG) antibody secreting cells in the intestine, spleen and PBMC of Gn pigs vaccinated with 1 oral and 2 IN doses of each vaccination regimen (HuNoV-HS66VLP+ISCOM or mLT) or controls……………………………………………………………..267
4.4 Cytokine concentrations in the serum of Gn pigs vaccinated with 1 oral And 2 IN doses of each vaccination regimen (HuNoV-HS66VLP+ISCOM or mLT) or controls……………………………………………………………268
4.5 Th1 (IFN-γ and IL-12), pro-inflammatory (IL-6), Th2 (IL-4) and Th2/T-reg (IL-10) cytokine concentrations in the intestinal contents of Gn pigs vaccinated with 1 oral and 2 IN doses of each vaccination regimen (HuNoV-HS66VLP+ISCOM or mLT) or controls……………………………………………………………..269
4.6 Mean numbers of Th1 (IFN-γ and IL-12) and pro-inflammatory (IL-6) cytokine secreting cells in intestine, spleen and PBMC of Gn pigs vaccinated with each vaccination regimen (HuNoV-HS66VLP+ISCOM or mLT) or controls……………………………..270
4.7 Mean numbers of Th2 (IL-4) and Th2-T-reg (IL-10) cytokine secreting cells in intestine, spleen and PBMC of Gn pigs vaccinated with each vaccination regimen (HuNoV-HS66VLP+ISCOM or mLT) or controls……………………………271
xxi
LIST OF ABREVIATIONS
Antigen-presenting cells (APCs) B cell receptor (BCR) Bovine enteric calicivirus (BEC) Bovine norovirus (BoNoV) Calicivirus (CV) Centers for disease control (CDC) Cesium chloride (CsCl) Cholera toxin (CT) Complementary RNA (cRNA) Dendritic cell (DC) Dimer IgA (dIgA) Double stranded RNA (dsRNA) E coli heat labile toxin (LT) Elongation factor (eLF) Enteric calicivirus (ECV) Enzyme-linked immunosorbent assay (ELISA) Feline calicivirus (FCV) Forkhead box P3 (FOX P3) Fucosil transferase (FUT) Gnotobiotic (Gn) Gram (g) Granulocyte/macrophage colony-stimulating factor (GM-CSF) Gut-associated lymphoid tissue (GALT) Hawaii virus (HV) High endothelial venules (HEV) Horseradish peroxidase (HRP) Hours (hrs) Human calicivirus (HuNoV) Human norovirus (HuNoV) Human rotavirus (HRV) Human sapovirus (HuSaV) Immuno-electron microscopy (IEM) Immunoglobuline (Ig) Immunoglobulin-like transcript (ILT) Immunostimulating complex (ISCOM) Interferon (IFN)
xxii Interferon alpha receptor (IFNAR) Intestinal content (IC) Intraepithelial lymphocytes (IELs) Intranasal (IN) Isolated lymphoid follicles (ILF) Jena virus (JV) Lamina propria (LP) Leeds virus (LE) Lewis (Le) Lineage marker (LIN) Lipopolysaccharide (LPS) Liter (L) Lordsdale virus (LDV) Lymphotoxin (LT) Major histocompatibility complex (MHC) Membranous cell (M cell) Mesenteric lymph nodes (MLN) Mexico virus (MX) Microgram (μg) Milligrams (mg) Molecular weight (MW) Monoclonal antibodie (MAb) Mucosa-associated lymphoid tissue (MALT) Mucosal addressin-cell adhesion molecule 1 (MadCAM-1) Muramyl dipeptide (MDP) Murine norovirus (MNV) Nanometer (nm) Nasopharynx-associated lymphoid tissue (NALT) Natural killer (NK) Nebraska (NB) Norovirus (NoV) Norwalk virus (NV) Nuclear factor inducing kinase (NIK) Nucleoside triphosphate (NTP) Open reading frame (ORF) Pathogen-associated molecular pattern (PAMP) Peanut agglutinin (PNA) Peripheral blood mononuclear cells (PBMCs) Peripheral lymph node addressin (PNAD) Poly(A)-binding protein (PABP) Poly(A)-binding protein (PABP) Polymerase chain reaction detectable units (pdu) Polymeric Ig receptor (pIgR) Porcine enteric calicivirus (PECV) Protein kinase A (PKA)
xxiii Rabbit hemorrhagic disease virus (RHDV) Real-time (rt) Recombinant bovine norovirus capsid antigen (rBoNoV) Recombinant Norwalk-virus (rNV) Recombinant virus like-particles (rVLP) Recombination-activating gene 2 (RAG2) Red blood cells (RBCs) Reverse transcription polymerase chain reaction (RT-PCR) RNA interference (RNAi) RNA-dependent RNA polymerase (RdRp) Rt (room temperature) San Miguel sea lion virus (SMSV) Sapovirus (SaV) Secretory component (SC) Secretory IgA (sIgA) Sensitive factor attachment protein receptor (SNARE) Signal transducer and activator of transcription 1 (STAT-1) Single stranded RNA (sRNA) Snow Mountain virus (SMV) South Hampton virus (SHV) T cell receptor (TCR) T helper (Th) T regulatory (T-reg) Tissue culture (TC) Toll-like receptor (TLR) Tumor necrosis factor (TNF) Venezuelan equine encephalitis (VEE) Venezuelan equine encephalitis replicon expression system (VRP-NV) Vesicular exanthema swine virus (VESV) Vesivirus (VSV) Virus protein genome linked (VPg) Virus structural protein 1 (VP1) Virus structural protein 2 (VP2) Wild-type (WT)
xxiv CHAPTER 1 LITERATURE REVIEW
ENTERIC CALICIVIRUSES AND IMMUNITY
1.1 Introduction and History
1.1.1 Norwalk virus discovery
In 1972, a 27 nm virus like-particle, referred to as Norwalk virus (NV), was observed by immuno-electron microscopy (IEM) in a fecal filtrate sample derived from an outbreak of gastroenteritis in an elementary school in Norwalk, Ohio, using a convalescent serum from a volunteer as the antibody source (203, 205). The fecal filtrates from this outbreak were used in volunteer studies during the early 1970s in an attempt to identify fecal suspensions that could cause infectious disease (90).
The first reported outbreak caused by NV took place in 1968 in an elementary school in Norwalk, Ohio and fifty-percent of the students and teachers developed acute gastrointestinal illness. The clinical symptoms occurred in the first 24 hours and were characterized by nausea, vomiting and abdominal cramps. These symptoms were characteristic of the “ winter vomiting disease” described in 1929 and had no defined etiological agent, since no bacteria could be isolated at the time (7). Well water was suspected of being the source of infection, but this was not confirmed (7).
1 1.1.2 Early volunteer studies
The first calicivirus (CV) volunteer studies began in 1972. These studies were
characterized by the fact that most adult volunteers had clinical symptoms after experimental exposure to NV, raising questions about the agent’s virulence or the lack of protection in the general population (90). Other studies revealed that the volunteers did
not develop clinical symptoms after re-challenge with the same homologous viral inoculum at 6-14 weeks after initial exposure (399). More quantitative methods were then applied to determine NV serum antibody titers in volunteers’ sera (117).
1.1.3 Recent volunteer studies
With the advent of new techniques such as the solid-phase immunoassays, the similarities between NV and other viral isolates from various outbreaks in different places such as Hawaii and Maryland were evaluated. In those studies, volunteers became ill after re-challenge with heterologous (from a different outbreak) isolates but not with homologous (NV) virus, and the reactivity between certain viral isolates with sera from volunteers previously infected with the same inoculum was confirmed by IEM (46). The relationship between high or low serum antibody levels and protective immunity against
NV was later determined (200). It became clear in those studies that preexisting serum antibody to NV was not associated with protection after multiple exposures and the existence of short-term immunity to the homologous virus was also reaffirmed (200,
253).
2 After 1990, new reagents such as recombinant virus-like particles (rVLPs) and
hyperimmune antisera (126) became available and new discoveries about cross-reactivity
and genetic determinants for susceptibility and/or resistance to CV were made (56, 126,
223, 224).
1.2 Classification
1.2.1 Taxonomy and genome organization
The Caliciviridae family is divided into four distinct genera: Norovirus,
Sapovirus, Lagovirus, and Vesivirus (40, 133, 255).
The noroviruses (NoVs) are classified into 5 genogroups (GI-V) and at least 27
genotypes, based on partial capsid, and regions A-D of the RNA dependent RNA
polymerase (RdRp) (102, 138). Strains of three genogroups (GI, GII, and GIV) are found
in humans (102). Porcine NoV strains are classified into three genotypes within GII
(GII.11, 18, and 19) (104, 358), and the bovine enteric caliciviruses (BECV) Jena and
Newbury agents form the third genogroup (226), with the Nebraska (NB) strain still
unclassified (226, 232, 335). The NV is the prototype for the GI human norovirus
(HuNoV) strains, and like the other viruses in the family, it was named after the location of the first outbreak in which it was first detected (202); however, this naming method
has become obsolete (133). The most recent proposed nomenclature for NoV and
sapoviruses (SaVs) is species infected/virus genus/virus name/strain designation/year of
isolation/country of isolation (20).
The SaVs are classified into 5 genogroups (GI-V), four of which infect humans
(GI, GII, GIV and GV) and one that infects animals (GIII), and at least 9 genotypes based
3 on sequences of the whole capsid protein gene and of the RdRp (105, 287). The prototype
Sapporo virus belongs to GI (194), and the porcine enteric calivirus (PECV)/Cowden
strain and other porcine SaVs constitute the third genogroup (143, 332).
The Lagovirus genus includes the rabbit hemorrhagic disease virus (RHDV) and
the European brown hare syndrome virus (EBHSV) that infect rabbits and hares (369).
The genus Vesivirus contains the prototype of the genus, Vesicular exanthema
virus of swine (VESV), the San Miguel Sea Lion virus (SMSV), feline calicivirus (FCV)
(39) and the canine calicivirus as a tentative member (369). Members from this genus
have been associated with infections in a wide range of hosts such as cats (310), dogs
(101), cattle (340), swine (42), skunks, mink (339), reptiles (29), marine mammals, fish,
amphibians (342), birds and primates (341), including humans (337).
In general, caliciviruses belonging to the same genus share a high degree of
genetic and antigenic relatedness, but have low sequence identity to members of other
genera (39).
The CV genome is composed of a single-stranded positive-sense RNA of
approximately 7.6 kb in length that encodes three open reading frames (ORFs). The
ORF1 encodes a polyprotein similar to the picornavirus 2C helicase, 3C protease, and the
3D, RdRp. However, the structural proteins in picornaviruses are encoded in the 5’ end of the genome and in the caliciviruses these genes are located in the 3’ portion of the genome (369). This major polyprotein is then cleaved by the viral protease resulting in nonstructural proteins required for RNA genome replication and generation of progeny virus. The ORF2 encodes the major capsid protein VP1, and ORF3 encodes the minor structural protein VP2 (202). The genomic organization between NoVs is generally
4 conserved among the different strains, and it is similar to the feline calicivirus (FCV) and
to the rabbit hemorrhagic disease virus (RHDV) genomes (202). The ORFs 1 and 3 of
NV and Snow Mountain virus (SMV) are in the same reading frame. This could be a consequence of a 17 nucleotide (nt) overlap between ORF 1 and ORF 2 that contains the
first AUG of ORF 2 and the stop codon of ORF 1, creating a –2 frameshift, and also of
the 1-nt overlap between the stop codon in ORF 2 and the start codon in ORF 3, bringing
the reading frame to the original position (202).
Other changes have also been detected between HuNoV and some animal CV
genomic organization (39). One of them is that the ORF 2 of the FCV, RHDV and also of
SMSV is longer than ORF 2 of NV and South Hampton virus (SHV) (202). The RHDV
genome has only two predicted ORFs because the capsid protein sequence is encoded
within ORF 1 (296), and another notable difference is that the FCV genome has –1
frameshift at ORFs 1 and 2 and 2 and 3 junctions, resulting in different reading frames for all three ORFs (138, 202).
The SaV genome is composed of only 2 ORFs (1 and 2). The ORF 1 encodes the polyprotein that has its sequence fused to the capsid protein resulting in a single large
polyprotein and ORF 2 that encodes for a small basic protein (138). The genome of the
porcine enteric calicivirus has 7,320 bp, excluding the 3’ poly (A) tail, and has similar
organization to the genome of the human SaVs (HuSaVs), being most closely related to
the HuSaVs than to other animal CVs (143).
The BEC genome is 7,453 bp long and has two ORFS (1 and 2). The ORF 1
encodes a large nonstructural polyprotein in continuity with the major capsid protein and
ORF 2 encodes for a small basic protein of 225 aa (226).
5 The complete NB virus genome sequence has only up to 26.7% identity with the genomes of other CV strains such as NV, and some vesivirus and lagovirus strains (335).
1.3 Viral structure
Cryo-electron microscopy structural studies of various CV have shown that their capsids exhibit a T=3 icosahedral symmetry consisting of 90 dimers of the virus structural protein 1 (VP1) (76, 123, 346). The viral capsid encloses the viral genomic
RNA that is approximately 8kb in size and is composed of either 2 or 3 major ORFs (76).
A virus protein genome linked (VPg) that is covalently linked to the 5’ end of the RNA genome and a 3’ end basic protein (VP2) are also present in the CV virions (138).
1.3.1 Structural proteins
-VP1. The VP1 is the major capsid protein of the members of the Caliciviridae family and is encoded by ORF 2; however, in some animal caliciviruses such as the
RHDV, the VP1 sequence is fused to ORF1. In the human caliciviruses NV and SHV it is composed of 530 aa and has an apparent molecular weight of 58K (138, 193). In some animal caliciviruses such as the RHDV, it has two additional amino acids at the N- terminus that during capsid maturation are removed by proteolytic cleavage yielding a smaller mature capsid protein (19). This single protein when expressed in insect cells infected with recombinant baculovirus, self-assembles into recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to the native NV
(193). Electron cryomicroscopy analysis of these rNV VLPs and computer image processing techniques have revealed that the capsid is composed of 90 dimers of the 58 K
6 protein that form a shell domain from which arch-like capsomeres protrude. The shell
domain consists of the N-terminal amino acids 1 to 250 and the remaining C-terminal
residues form the arches (309). Through antigenic mapping of the rNV VLPs using
monoclonal antibodies (MAb), discontinuous epitopes were revealed, demonstrating that
the rNV capsid protein is at least partially folded, while other MAbs recognized continuous epitopes. Most MAbs map to the C-terminal half of the protein, suggesting
that this region may contain the immunodominant epitopes (158). Amino acid identities
between strains of distinct NoV genogroups vary from 37-44% and strains within the
same genogroup share 61-100% homology. The greatest sequence divergence between
strains resides within the central variable region (132).
Comparative analysis of the structure of the recombinant capsid of viruses
representative of NoV GII, SaV and Vesivirus (VSV) showed that all capsids are
composed by 90 dimers of the capsid protein arranged on a T=3 icosahedral lattice with
each subunit consisting of a shell domain and a protrusion domain. The most distal
(protrusion 2) domain is the most variable domain in size and shape confirming the
sequence variability (72). More recent X-ray structural analysis revealed that host range
may be a reflection of the unique capsid structure of some viruses. The VSV capsid
structure, represented by the SMSV, has the same architectural organization as the
recombinant NV capsid. However, it has unique interdomain flexibility that is important
for the capsid-related functions and also plays a role in strain diversity. In this virus, the
neutralization epitopes map to three hypervariable loops surrounding a region in the distal
portion of the protruding domain encompassing a region that exhibits host-specific
conservation (71).
7 -VP2. This small basic protein is encoded by ORF3. It has a predicted molecular weight of approximately 22K, and is composed of 212 aa residues. The basic nature of this protein suggests that it may be involved in nucleic acid binding and, since the existence of ORF3 is conserved among the CVs, it is presumed that this protein may play a role in viral replication or assembly (108). Recombinant baculovirus expressing either only
ORF2 or both ORFs 2 and 3 assemble into rNV VLPs (123, 135, 148), showing that the
ORF3 protein is not required for the rVLP formation; however, it may be important in regulating the expression and stability of the viral capsid protein VP1 (43). It was recently demonstrated that FCV VP2 is essential for the production of infectious virions
(346), suggesting that the function of this protein is still poorly understood.
1.3.2 Nonstructural proteins
Most of the studies of nonstructural proteins are based on the similarities with
picornavirus nonstructural proteins, with the human CV prototype strain NV (276), and
also on NoV protein expression and in vitro mapping studies (36, 47).
The NV nonstructural polyprotein encoded by ORF1 has 1,789 amino acids and
after cleavage, results in an NTPase (p41), a 3C-like protease (3Clpro), an RdRp and a
VPg from N to C terminus in this order: p48-p41-p20-VPg-3Clpro-RdRp (138).
-p48. This protein consists of the N-terminal 398 aa and may play a role in viral
replication because it has no significant sequence similarity to any protein in the public
databases. The H-Box/NC sequence motifs have been found in p48 suggesting a function
in the control of cell proliferation (177). After transfection in COS-7 cells, P48 localizes
to the intracellular vesicle when expressed transiently as an EYFP fusion protein. When
8 cells coexpressed p48 and the vesicular stomatitis virus G glycoprotein its expression on
the cell surface was inhibited, suggesting that p48 may disrupt intracellular protein
trafficking (100).
-p41. This protein is a putative superfamily 3 helicase with NTPase activity (76, 138,
306). No function has been identified for this CV protein; however, it has been
demonstrated in vitro that the SHV p41 protein is a nucleic acid-independent nucleoside
triphosphatase (306).
-(3Clpro). This 3C-like proteinase belongs to a group of trypsin-like proteases encoded by the super-family of positive-strand RNA viruses (92), and is responsible for the processing of the ORF1 polyprotein into the nonstructural proteins (157, 225). Proteolytic processing of NV occurs at glutamine-glycine or glutamate-glycine/alanine junctions within the mature polypeptides (47). It has been characterized as a chymotrypsin-like protease in which the His-30 and Cys-139 residues form the catalytic site (225, 343).
Cysteine is the nucleophilic residue in the proteinase, located at C 1238 for SV (225) and
C1239 for NV (157). The MD145-12 strain proteinase has been expressed in bacteria and
could not only cleave its own mapped cleavage sites in trans but also mediate trans
cleavage of the NV ORF1 polyprotein in similar fashion. A time course analysis showed the formation of stable precursors p20VPg and ProPol, and less stable p20VpgProPol, p20VpgPro, and VpgPro during the proteolytic processing (37). It is speculated that the
calicivirus proteinase may also play a role in the host cellular translation by cleavage of
poly(A)-binding protein (PABP) (219).
-p13 (VPg). This protein is covalently linked to the 5’ end of the CV positive-strand
RNA genome. Earlier studies showed that if VPg was removed from the FCV mRNA, the
9 translation of the genomic RNA was significantly decreased (166), suggesting that VPg could play a role in protein synthesis. The NV VPg also interacts with the initiation factor elF3 and with other initiation factors (83). The FCV and Lordsdale (LDV) VPg binds directly to elF4E and this interaction is necessary for translation of the calicivirus genome in vitro (125), supporting its role in translation initiation complex recruitment. However, calicivirus strains may differ in their requirements for translation because, although the murine norovirus (MNV) VPg interacts directly with elF4E, this initiation factor is not required for MNV RNA translation (68).
-p22 (GI) or p20 (GII). The function of this protein has not yet been identified.
-RNA-dependent RNA-polymerase (RdRp). This CV protein is encoded by the 3D region of the genome. It has a conserved aa motif, glycine-aspartic acid-aspartic acid
(GDD) that is also present in the active site of various viral RdRp, and it is related to the picornavirus 3D polymerase (214). The RHDV Pol structure has a “ right-hand” conformation (278) characteristic of the positive-strand RNA virus RdRps. This RdRp is crucial for the synthesis of both plus- and minus-strand RNA molecules, since in NV the genomic RNA serves as a template for the minus-strand RNA synthesis, that then serves as template for the synthesis of the progeny genome plus-strand RNA molecules. The
RHDV and the FCV RdRp have been characterized as enzymatically active proteins, and both enzymes can synthesize RNA from a polymeric template in vitro in the absence of an exogenous primer (230, 388). Recently, it was shown that the completely processed
Pol of NoV RdRp, expressed in bacteria, was enzymatically active and that the ProPol precursor of the GII MD145-12 is an active RdRp (36, 278), suggesting that the precursor
ProPol may be the actual functional enzyme during NoV replication.
10 1.3.3 Virus-like particles (VLPs)
When only the gene encoding the major capsid protein (ORF2) or when both
genes encoding the capsid protein (ORF2) plus the gene encoding the small basic
protein (ORF3) are transferred into a recombinant baculovirus and the proteins are
expressed in insect cells, they self-assemble into rVLPs, producing high yields of CV
particles.
Those particles are devoid of any nucleic acid but they are morphologically and
antigenically similar to native virus particles (130). Although the inclusion of the small
basic protein is not a requirement for the assembly of the rVLPs, some studies have
shown that this protein may provide more stability to these particles.
According to X-ray studies of the NV rVLPs, the shell domain seems to have all
the requirements for the initiation of capsid assembly, and the contact between the
protruding domain of each protein helps to stabilize the capsid, and may also be
important in determining the capsid size (44). As an alternative to the baculovirus-
expression system, the rVLPs can also be produced using mammalian-cell based protein
production systems (367).
Because HuNoV do not grow in cell culture, these VLPs have been important
models for structural and viral-cell interaction studies of the CV (229, 381, 391) and
have also played a major role in the generation of new reagents because they can be
used as antigens in enzyme-linked immunosorbent assays (ELISAs) and also to
immunize animals for the production of antisera (69, 148).
The use of rVLPs as carriers for foreign epitopes has also been evaluated, and
when the N-terminal 30 aa of the capsid protein of a RHDV strain was substituted by a
11 few residues containing an epitope of the capsid protein of the bluetongue virus, the
resulting fusion protein self-assembled into rVLPS, retaining the antigenicity of both
viruses (273).
Immunization of different animals with NoVs and SaVs rVLPs has been
evaluated (146, 302, 374). High titers of specific neutralizing antibodies against
PECV/Cowden were observed in guinea pigs inoculated with PECV rVLPs (147).
The viral capsid protein of the SaV Sapporo/82 has also been expressed in
baculovirus (283), and the expression of the viral capsid protein of two SaV strains
(Hou/90 and Hou/86) has also resulted in rVLPs. Different constructs were compared
and only the capsid protein with the tri-peptide MEG resulted in assembly of rVLPS.
Some failed to produce the viral capsid and the others that expressed capsid protein did
not assemble into rVLPs (195).
In conclusion, rVLPs are immunogenic eliciting both serum and mucosal immune responses (24, 26), representing an effective form of subunit vaccine that mimics the viral structure without the infectious genetic material, constituting an important alternative to live vaccines without the risk of viral reversion, recombination and/or re- assortment. A more detailed discussion about the use of rVLPs as an immunization alternative against HuNoV will be provided in the “vaccines against caliciviruses” section of this literature review.
1.4 General biological features
The HuNoVs have a “fuzzy” surface by negative-stain electron-microscopy, although no indentations are present on their capsid surface. They have a buoyant
12 density, determined by the rVLPS distribution in Cesium Chloride (CsCl) gradient that
ranges from 1.36 to 1.41 g/cm3, except for the less dense SMV that has a buoyant density
of 1.33 in CsCl gradient (138).
The HuSaV, when viewed by EM, often display the CV characteristic “cuplike”
morphology. They have a buoyant density that ranges from 1.37 to 1.38 g/cm3 in CsCl gradient (138).
1.4.1 Viral stability and infectivity
The HuNoVs are generally very stable in the environment, which greatly contributes to viral spread and disease (138). Due to their high infectivity, great stability and disease impact, these agents have been recently classified as a group B biodefense pathogens (180).
Studies showed that NV particles remained infectious for volunteers after exposure to pH 2.7 for 3 hrs at room temperature (Rt). The NV particles were also resistant to 20% ether at 4°C for 18 hs and to incubation at 60°C for 30 min (202).
The chlorine concentration (from 0.5 to 1 mg/L) normally used in the treatment of the water supply system is not sufficient to inactivate NV; however, NV is inactivated with 10 mg/L of chlorine (210). This concentration is usually used when contamination has been detected in the water supplies. Because FCV has similar physicochemical properties and genome organization to those of NV, it has been used as a surrogate for inactivation studies. In one study, the low-pressure UV radiation (254 nm) dose required to inactivate 99% of the FCV particles in buffered water was 16 mJ/cm2 (371). Therefore,
the use of UV should be considered for a HuCV inactivation method. The NV
13 concentration in raw sewage has also been evaluated. Before treatment it was
approximately 105 pcr detectable units (pdu) per liter and after treatment, 103 pdu/L of
HuNoV were still detectable (378).
It has been reported that a significant amount of FCV (5 to 10 logs) can be transferred from contaminated surfaces such as cutting boards, door knobs, and lettuce to foodhandler gloves (299), and that NV and FCV were still detectable on the surface of lettuce leaves for up to a week after their contact with virus seeded stainless steel surface
(93), constituting a potential source of food related outbreaks. Recently, the virucidal efficiency of different chemicals against NV and FCV has been tested. The R-82, a quaternary ammonium compound, completely inactivated FCV after 10 minutes of contact with the contaminated surfaces (196). The virucidal Metricide, an activated dialdehyde-based product, inactivated 99.99% of the FCV on fabrics and carpet surfaces within 10 min of exposure (241).
1.4.2 In vitro viral replication
The study of HuNoV has been greatly hampered by the fact that these fastidious viruses have not yet been adapted to routine in vitro cultivation methods, although several attempts have been made (94) and one recent report of HuNoV growth in an organoid model of human small intestinal epithelium suggests some success (356). In these efforts different cell lines, such as Caco-2, CCD-18, HeLa, MA104, MDBK, among others, and different culture supplements (insulin, DMSO, butyric acid) have been used in trying to adapt these viruses to cell culture, but without any success. Currently, the only CV that have successfully grown in cell culture are: one SaV (PECV/Cowden) (111, 298), VSVs
14 (canine, chicken and feline), the murine norovirus 1 (MNV-1) and only recently, HuNoV
GI and GII replication was detected in a 3-dimensional organoid model of human small intestinal epithelium. After growing the cells on porous collagen-I coated microcarrier beads under conditions of physiological fluid shear conditions in rotating wall vessel bioreactors, the desired level of cell differentiation was achieved. The final model resembled the morphologic and physiologic function of in vivo tissues. The GII and GI
NoV-positive stool samples from cruise ship outbreaks were used to infect the created tissue, and after 24-66 hrs post-infection, particles that resembled NoV particles accumulated within the tissue and were detected by transmission electron microscopy.
Infection was confirmed by RT-PCR with posterior sequencing and also by in Situ hybridization, and cytopatic effect. The GI and II norovirus RNAs were detected in each of the 5 passages performed (356).
The PECV/Cowden was first adapted to cell culture, in our lab, after supplementation with intestinal content (IC) fluids from uninfected gnotobiotic (Gn) pigs in the culture medium (111, 298). A later study reported that the bile acids present in the
IC act as inducers of a protein kinase A signaling pathway that seems to be essential for the Cowden strain growth in cultured cells (64, 66).
The MNV-1 infects macrophage-like cells in vivo and it replicates in cultured primary dendritic cells and macrophages in a signal transducer and activator of transcription 1 (STAT-1) regulated fashion (397), providing important information on norovirus biology and interaction with the host immune system.
15 Many VSVs have also been grown in cell culture such as the canine CV, originally isolated from feces of a dog (330), a chicken enteric CV that required addition of trypsin to the growth media (80), and also the FCV (234, 235) that has been used as an important surrogate for the study of caliciviruses.
This recent success in culturing HuNoV, together with all the other previous attempts, suggest that many factors such as cellular differentiation stage, tissue architecture, gut microenvironment, and antigen expression, may play a role in HuNoV entry, binding and/or internalization into the intestinal epithelial cells, making the routine in vitro cultivation of these agents still a challenge.
1.4.3 Caliciviruses infectious clones
Infectious clones from many CV strains have been developed using different cloning strategies and viral expression methods (18, 65, 67). The FCV RNA transcripts, derived from a cDNA clone from the Urbana strain, were infectious after transfection into feline kidney cells. In this system the VPg that is essential for Wt-FCV was not required for successful virus recovery. However, the addition of a cap structure analogue, during in vitro transcription of the RNA, was necessary for virus recovery (345).
The PECV RNA transcripts, derived from a full-length cDNA clone of the PECV genome, were infectious after transfection into a continuous porcine kidney cell line
(LLC-PK). Bile acids were a requirement for viral recovery after transfection, and the recovered virus was infectious after oral inoculation of gnotobiotic pigs (65).
16 A full-length infectious clone of the RHDV strain JX/CHA/97 has also been produced, and the RNA transcripts derived from this clone were replication competent when transfected into rabbit kidney (RK-13) cells (227).
Mammalian cell-based systems have also been established for the study of
HuNoV replication. In one of these systems, the NV viral RNA transcription was driven by T7 RNA Pol produced by the recombinant vaccinia virus strain MVA. The RNA was then packaged into virus particles in human embryonic kidney cells (HEK293T cells) cytoplasm. The expressed genomic RNA was replication efficient, the subgenomic RNA
was transcribed from genomic RNA using NV nonstructural protein expressed from
genomic RNA and was then translated into NV capsid protein VP1, making this cell-
based system useful for the study of HuNoV replication and for the development of
antiviral drugs (18). In another study, the NV RNA was expressed by the vaccinia virus
MVA/T7 system, and the genomic RNA replicated in 293T cells. The sub-genomic RNA
was transcribed from genomic RNA using NV nonstructural proteins expressed from the
genomic RNA and was then translated into the VP1 NV capsid protein. Viral genomic
RNA was subsequently packaged into viral capsids and retained the buoyant
characteristics of the original NV isolated from human fecal samples. A stable NV RNA
replicon system that can replicate in human (Huh-7) and hamster (BHK21) cell lines
without the need for a helper virus has also been recently generated. In this system, when
the cells were treated with interferon-alpha (IFN-α), prior to transfection, NV replication
was significantly reduced (67), providing new insights on viral-host interactions.
17 The engineering of infectious clones from different strains has been crucial for the
study of calicivirus biological features, replication, and pathogenesis, however there is
still much to learn about cell-virus interaction and the requirements for enteric HuNoV
attachment, entry and replication in the intestinal cells.
1.5 Diagnosis
After the initial outbreaks caused by NV, the first clinical and epidemiological
characteristics of NoV illness consisted of nausea, vomiting and abdominal cramps,
although diarrhea in some epidemics was more common. If bacterial or parasitic agents were not detected in those patients experiencing these symptoms, NV was considered the probable cause of disease (1, 7). The duration of illness varies from 12-60 hrs with an incubation period of 24 to 48 hrs (138).
More recently, with the identification of more viral strains, resulting in the production of new antibodies and RT-PCR primers, more sensitive and specific caliciviruses detection techniques are being developed. However, the inability to routinely propagate these viruses using monolayer cell cultures has been an obstacle for rapid and reliable laboratory diagnosis that usually requires more than one technique to be successfully achieved (20).
-Electron microscopy. The use of electron microscopy as a diagnostic tool is still valuable because it is rapid for small numbers of samples and it does not require extensive prior sample preparation. However, it requires the maintenance of expensive equipment and facilities and trained personnel. One disadvantage of this technique is the requirement for a large concentration of particles in the sample (about 106 particles)
18 (202). The sensitivity and specificity of electron microscopy may be enhanced by IEM, whereby virus specific antibody is used to agglutinate virus particles making the visualization and identification of the viral particles easier (205).
-Reverse transcriptase-polymerase chain reaction (RT-PCR). The RT-PCR is the main molecular identification tool in the detection and identification of HuNoVs (12, 13,
21). Many primers have been designed based on the highly conserved regions of the
RNA genome such as the polymerase region or the ORF1-ORF2 junction regions.
However, more recently, with the identification of recombinant viruses and multiple
mutations mainly in the capsid region of the genome (281, 384), more attention is given
to the more variable regions of the genome such as the capsid gene for genotyping of
viruses (382). During an outbreak, choosing the right primers is essential due to the high
genetic variability among the different circulating strains. Before the CV can be detected
by RT-PCR a successful RNA extraction is essential. The use of internal controls is also
recommended to avoid false negative results due to the presence of PCR inhibitors that
are usually present in the biological samples such as fecal and vomit samples (20).
a) RNA extraction methods.
This is the first, and the most critical, step of sample processing before RT-
PCR detection of viral RNA. Many different methods of extraction have been tested since
the discovery of “small round structured viruses”. In one study, four methods of RNA
extraction (using Chelex-100, Sephadex G200, Guanidium thiocyanate or polyethylene
glycol precipitation followed by phenol/chloroform extraction) were compared for their
efficiency of viral RNA recovery and removal of PCR inhibitors.
19 The most sensitive methods used the metal chelating agent Chelex-100 and or the
Sephadex G200 column chromatography and these methods detected a 10-4 dilution of a positive “small round structured virus” sample. The Guanidium thiocyanate/silica method had intermediate sensitivity while successfully removing inhibitors from previously negative samples that were spiked with SRSV(150).
In another study, samples were seeded with canine CV and the sensitivity of two
RNA extraction methods (TRIzol and RNA Mini Kit Quiagen) was compared for detection of NoVs in dairy and vegetable samples. The TRIzol extraction method, after
previous virus concentration using polyethylene glycol plus NaCl ultracentrifugation and
ultrafiltration was the most efficient method for the extraction of viruses in dairy samples.
The Rneasy-Mini Kit procedure, preceded by ultracentrifugation of the samples was the
most sensitive for detection in vegetables (324).
b) Primers. Since HuSaVs are genetically more similar to animal CV than to other
human calicivirus (HuCV), the use of primers that are both broadly reactive and
reasonably sensitive for screening of a possible HuCV infection is very important. The
first primers designed based on the conserved RNA polymerase region of HuCV
genomes detected strains from both SaV and NoV genera, but with low sensitivity of
detection for some known HuCV strains (192). A primer pair (p289/290) that detects both
NoV and SaV strains has been designed and its specificity and sensitivity evaluated
(191). The primers detected prototype samples from NoVs (NV, SMV, Hawaii and
Mexico viruses) and SaVs (Sapporo/82, Hou/86, Hou/90 and Lon/92), as well as other
circulating NoV and SaV strains, and proved to be very useful during HuCV outbreaks,
clinical and environmental investigations. The primers were also able to amplify BECs
20 and some NB-BEC strains (336), and also non-enteric mink caliciviruses (MCV) that
were later found to be VSVs as well as mink SaVs (144). The disadvantage of this primer
pair is that it can also detect rotaviruses non-specifically (237).
c) RT-PCR internal controls. The addition of viral RNA sequences as internal standard
controls in RT-PCR assays is an important tool for CV detection in samples that could
potentially contain inhibitors (20). Amplification inhibition seems to be caused by an
inbalance between the amount of inhibitors, the amount of virus present in the sample, and the efficiency of the RNA extraction method coupled with its ability to remove
inhibitors.
Different internal controls have been designed but problems have been encountered such as similar size to the target product making it difficult to distinguish between the two during gel electrophoresis and also the lack of versatility due to amplification by primers that are not broadly reactive and are relatively common.
An internal standard was created by cloning a 319 nt sequence of the NV
polymerase gene containing a 156 nt cDNA insert, resulting in a 475 nt RT-PCR product
that was amplified by the broadly reactive primer-pair 289/290 (191) that can detect both
norovirus and sapovirus HuCV strains (99).
-Real-time PCR. Real-time (rt) RT-PCR assays were developed for detection and quantification of HuNoVs GI and GII in biological samples (265, 313) providing a more sensitive, rapid and quantitative method for HuCV detection. In one study, a single tube
SYBR green rt RT-PCR was developed to detect and semi-quantify GI NoVs in stool samples. The method amplified NV RNA to as high as 6.16 x 1010 g-1 of stool (313).
Another single tube rt PCR assay detected 79% of the CV-positive samples derived from
21 outbreaks and sporadic cases in Sweden during 1997-2004, that had been previously
detected by EM. The CV cDNA was initially amplified using primers and Taq Man
probes targeting the ORF1-ORF2 junction region, followed by DNA amplification by the
rt technology (265).
-Microwell hybridization assays. This methodology uses probes that are coated onto
microwell plates and after the denatured RT-PCR amplicons, that were previously
amplified with a biotin-labeled primer are added to the wells the reaction is visualized by
the addition of streptavidin conjugated to the enzyme horseradish peroxidase (HRP) and
developed by the addition of the substrate (350). Another variation of the assay uses
streptavidin to coat the microwells and after the PCR products are added in the presence
of a binding buffer, digoxigenin-labeled probes are added and binding to the amplicons is
revealed by an anti-digoxigenin antibody conjugated with alkaline phosphatase followed
by the substrate (254). This assay has high senstitivity and it is useful to confirm RT-PCR
products. It is rapid to perform and multiple samples can be tested in a single plate. A
microwell hybridization assay was developed in our lab to confirm porcine NoV and SaV
RT-PCR amplicons. In that work the 5’ end of the forward NoV primers PNV7, PEC66,
PEC68, PSV6 and PSV11 that target the RdRp region of porcine NoVs and SaVs were biotin-labeled and the microwells were coated with probes that target the porcine NoVs
GII.11, GII.18 and GII.10 and the porcine SaVs GIII.1, GIII.2? and other SaV that may represent other three different genogroups of SaVs. The hybridization assay had similar or higher sensitivity (1-8-fold) than agarose gel electrophoresis depending on the probes used and the cut-off value for each assay (383).
22 A similar assay was also developed in our lab for confirmation of HuNoV amplicons
using Mon431-biotin labeled primer and probes specific for HuNoV GII-4 strains (69).
-Immunoassays. Most immunoassays that are routinely used for detection of HuNoVs
use hyperimmune antisera prepared against rVLPs. These assays are usually sensitive and
have high specificity to the immunizing rVLPs. The Escherichia coli expression system has been used to produce HuNoV capsid proteins for the generation of MAbs against
HuNoV (407). Some of these MAbs have broad reactivity detecting both HuNoV GI and
GII strains (212, 406), being very useful in ELISAs for detection of HuNoV antigen in biological specimens derived from outbreaks.
Commercial ELISAs are also available. Two ELISA kits, the SRSV (II)-AD
(Denka Seiken Co. Ltd., Tokyo, Japan) and IDEIA NLV (DakoCytomation Ltd., Ely,
United Kindom) were evalulated for routine diagnosis using a panel of GI and GII
HuNoV- positive stool samples. The Denka kit had >70% sensitivity for detection of 10 of the 14 HuNoV genetic subgroups, but a specificity of only 69%, and it also reacted with samples containing HuSaVs. The Dako kit had a low sensitivity (<30% for 6 GII subgroups) but a 100% specificity. Therefore none of the two kits have the combined specificity and sensitivity to substitute RT-PCR for HuNoV detection (59).
1.6 Animal caliciviruses
1.6.1 Porcine sapovirus
The PECV/Cowden is classified as SaV GIII (143) and it was first observed in our lab during examination of diarrheic stool samples of pigs from the U.S (326). The
PECV/Cowden is the only enteric calicivirus that has been successfully adapted to the
23 LLC-PK line with supplementation of IC fluids from uninfected Gn pigs in the culture medium (110, 298). The adaptation of PECV to cell culture provided more detailed information on the relationship between cell-signal transduction induced by IC and its effect on growth of PECV/Cowden in LLC-PK cells, indicating that bile acids are the active factors in the IC for PECV replication and that they induce the increase of cyclic
AMP concentrations in LLC-PK cells (64). This increase in cyclic AMP was later linked to the down-regulation of IFN mediated by the inhibition of activation of STAT1 that constitutes a key element in type I (IFN-α/β) and type II (IFN-γ) IFN responses. The
STAT1 is activated by phosphorylation after IFN-binding to their receptors triggering a signaling pathway that ultimately results in an antiviral state (64, 66).
The complete genome of both the tissue culture (TC) adapted PECV/Cowden
(TC-PECV) and the wild-type (WT) PECV/Cowden has been sequenced and sequence comparisons revealed that the TC-PECV/Cowden has one silent mutation in its protease, two amino acid changes and a silent mutation in its RNA polymerase, and five nucleotide substitutions in its capsid that give rise to one distant and three clustered amino acid changes and a silent mutation (143).
After experimental inoculation of Gn pigs, wild-type (WT) Cowden PECV caused mild to severe diarrhea in inoculated pigs (112). Villous atrophy was found in the duodenum and/or jejunum of the Gn pigs. In a later study, the pathogenesis of tissue culture adapted and WT-PECV/Cowden in Gn pigs was compared and it was observed that the TC-PECV/Cowden did not induce diarrhea in the orally inoculated Gn pigs, and only mild or no villous atrophy was observed in the small intestine.
24 The WT-PECV however, induced diarrhea in all orally or intravenously
inoculated Gn pigs and moderate to severe intestinal villous atrophy was detected.
Viremia was also observed following PECV infection (145).
In a more recent study, porcine GIII SaVs were detected by RT-PCR and
microwell hybridization assays, with SaV-specific primers and probes, in 62% (389/621)
of pigs from seven U.S swine farms and one slaughterhouse. A higher prevalence of
porcine SaVs was detected among postweaning pigs (83%) when compared to sows
(71%), finisher pigs (64%), and nursing pigs (21%). Among the postweaning pigs, the
SaV prevalence was higher in diarrheic pigs (100%) than in normal pigs (83%),
suggesting a role for GIII SaV in post-weaning pig diarrhea (385). However, porcine
SaVs that had between 76 and 93% nucleotide identity with the PECV/Cowden strain were detected at similar frequencies among fecal samples from Venezuelan piglets with or without diarrhea (252).
1.6.2 Porcine norovirus
In swine, NoV sequences were first detected in the caecum contents of two healthy pigs that lived in close contact with humans in Japan (358). These two strains were later classified into a new genetic subgroup within GII NoVs, based on the amino acid sequences of their entire capsid protein (359).
Genetically diverse porcine NoVs, that were classified into three genotypes within
NoV GII (GII.11, 18, and 19) were recently detected in our lab, with the GII-18 being genetically and antigenically most closely related to human GII NoVs. The GII-18 porcine NoVs also replicated in Gn pigs after experimental inoculation with viral
25 shedding being detected by RT-PCR and IEM. A potential NoV recombinant, resulting from homologous recombination within the highly conserved motif in the RdRp-capsid junction of different genotypes within the same NoV genogroup, was also identified in this study (384), raising concerns about the emergence of new human NoV or porcine/human GII recombinants.
In the same prevalence study in which researchers detected porcine SaVs in fecal samples from 7 US farms and 1 slaughterhouse, porcine GII NoVs were detected only in samples from finisher pigs with prevalence rates ranging from 3-40% among NoV- positive farms, with an overall prevalence of 20%. No obvious clinical signs of infection were observed and co-infection of pigs by NoVs and SaVs was detected in 27% of 188 finisher pigs (385).
The NoV antibody prevalence among domestic pigs was investigated among serum samples from the US and Japan. Seventy-one percent of pig serum samples from the US and 36% from Japan reacted with the recombinant capsid antigens of a prototype strain of swine GII NoV (SW918 strain). Sixty-three percent of the serum samples from pigs in the US were also positive for NV (GI) and 52% for Hawaii virus (GII), showing that NoV infections are common among domestic pigs in different parts of the world, suggesting that the swine GII NoV strain is antigenically similar to the human GII NoV strains (104).
1.6.3 Bovine norovirus and other enteric caliciviruses: NB strain
Calicivirus-like particles were initialy observed in the feces of calves from the
United Kingdom and Germany. These viruses, named Newbury agents 1 and 2 and Jena
26 virus, respectively (82, 142) are closely related to the GI HuNoV (82, 226), and were later classified into a new genogroup (GIII) within the NoV genus (226, 290). These viruses replicate in the enterocytes of the upper portion of the small intestine and cause diarrhea in experimentally inoculated calves and have also been associated with diarrhea outbreaks in calves (55). During a study that investigated the presence of NoVs in diarrheic fecal samples from calves in different dairy farms in the states of Michigan and
Wisconsin, strains from both BEC subgroups, Jena and Newbury agent 2 were detected in samples that originated from both states (396).
Another BEC, the NB strain was originally detected in fecal samples from calves in the US (335). Sequence analysis of its complete RNA genome revealed that its size and genomic organization are similar to strains in the Sapovirus and Lagovirus genera
(335), indicating that these agents could represent a new genus within the Caliciviridae
family. The NB virus caused diarrhea and lesions only in the duodenum and jejunum of
experimentally inoculated Gn calves (335). In a study conducted in our lab, samples
from two commercial Ohio veal calf heards were tested for the presence of enteric
caliciviruses using six different RT-PCR primer sets targeting the RdRp region of NoV-
GIII, NoV-like BECs, BEC-NB-like, and NB-related BECs. Caliciviruses were found in
three of four and four of four veal heards, respectively, and sequence analysis showed
that two BECs designated CV95-OH and CV186-OH were most closely related to the
BEC Jena and the Newbury agent-2 strains. After new primers were designed based on
the sequence of these strains, 72% of young veal calf fecal samples were positive for
BECs (336).
27 The complete capsid gene sequences of 21 BECVs revealed that 15 BECVs belonged to NoV genogroup III genotype 2 (GIII/2) and were genetically distinct from
HuNoV GI and GII. The capsid sequences of 6 BECV strains were similar to the NB strain. A potential recombinant between genotypes 1 and 2 strains of NoV GIII was also identified (155), and together with the sequencing data from the genome of another chimeric BEC that had a polymerase gene similar to the Jena virus and the capsid and
ORF3 like the Newbury 2 agent, the data suggest that coinfections with distinct BECV genotypes may occur and could play a role in diarrhea outbreaks among calves (289).
1.6.4 Murine norovirus
The MNV-1 was initially described in severely immunocompromised mice that lacked the recombination-activating gene 2 (RAG2) and STAT-1 (RAG2/STAT1-/-). The
MNV-1 could be passaged by intracranial inoculation and it was associated with encephalitis, vasculitis, meningitis, hepatitis, and pneumonia, with the mice eventually succumbing to the disease. However, in WT mice, no clinical signs or tissue pathology was detected and no viral RNA could be detected in any tissue by 3 days after inoculation.
The MNV-1 was more virulent in mice lacking both alpha/beta interferon (IFN-
α/β) and the IFN-γ receptors than in wild-type mice. When RAG1-/-, RAG2-/- or wild- type mice were infected by the peroral, intracranial, or intranasal (IN) routes, the RAG-/- mice were persistently infected, with high levels of MNV-1 RNA being detected in their feces, and also in their lungs, spleen, brain, and jejunum. The MNV-1 infection was also lethal in STAT1-only deficient mice. Therefore, STAT1, and the innate but not the
28 adaptive immune response, seemed to be essential for protecting the mice against lethal
MNV-1 infection. However, the presence of high levels of viral RNA in multiple organs
and in the feces of RAG-/- mice for up to 90 days after peroral inoculation, and not in the
wild-type mice, indicates that the adaptive immune responses may be necessary for the
containment and clearance of MNV-1 infection (207).
The MNV-1 replicates in different murine macrophage and dendritic cell lines,
and immunohistochemical staining of tissue sections from STAT1-/- mice revealed
macrophage-like and dendritic-like stained cells in the spleen, demonstrating its tropism
for cells of the hematopoietic lineage (397). Sequence data from the MNV-1 genome revealed that it is composed of three ORFs similar to the NoVs and vesiviruses, with
ORF1 encoding for a polyprotein containing 2C helicase, 3C protease, and 3D
polymerase motifs. The ORF2 encodes a capsid protein and ORF3 encodes a putative
small basic protein. Phylogenetic analysis based on the capsid protein sequences and viral genome demonstrated that the MNV-1 constituted a new NoV genogroup (GV) (207).
Mice from different breeds, such as adult 129 and juvenile CD1 that were inoculated with four MNV strains (1-4) did not show any clinical signs after inoculation (173). The MNV circulates among research mice, as shown by a seroprevalence study in which 22.1% of the mice serum samples tested were positive (173, 207).
29 1.7 Pathogenesis
1.7.1 Clinical features
Since its first reported outbreak in 1968, NV illness, initially described as “winter vomiting disease”, has been characterized by nausea, vomiting and abdominal cramps, and symptoms usually persist for 12-48 hours (7).
Transient malabsorption of fat, D-xylose, and lactose, and delayed digestion have
been observed in NV infected volunteers. This delay in gastric function could be
associated with the nausea and vomiting experienced by some patients infected with NoV
(202).
Other clinical symptoms, such as myalgia, headache, and chills have also been reported, lasting for 1-3 days (204). One of the most common complications of the
disease, especially in young children and elderly persons, is dehydration secondary to the
profuse diarrhea. Asymptomatic infection is also very common and it may be important
for HuNoV transmission from person-to-person (54). Characteristic jejunal lesions have
also been detected in volunteers that did not show any gastrointestinal symptoms (202).
The main clinical symptoms experienced by patients with SaV infection are the
same as those that afflict NoV patients. However, in young children and adults, diarrhea
seems to be the predominant sign during SaV infections (199, 294) in contrast to
vomiting in HuNoV infections. Headache, fever and myalgia are also frequent among
SaV-infected patients (199, 316).
30 1.7.2 Incubation period and viral shedding
The NV volunteer studies show that the incubation period for NV disease
development ranges from 10 to 51 hrs with a mean of 24 hrs. Virus shedding can be
initially detected by IEM during the onset of symptoms and for about 72 hrs after the
initial symptoms. Shedding has been detected by RT-PCR for up to 7 days after the onset
of symptoms in healthy adults (202).
Long-term HuNoV excretion is common in immunocompromised patients. A
child with cartilage hair hypoplasia had NoV gastroenteritis for 4 months and after an
interval of 5 months, the patient developed persistent NoV gastroenteritis that lasted for 6 more months, with the recombinant GII.3 HuNoV (ARG310/1999/US) being detected in the feces for 156 days (116). Another study showed that caliciviruses were detected more frequently in stools from HIV infected children than from HIV-negative children (51% versus 24%), suggesting that caliciviruses may be an opportunistic agent in HIV-positive children (319).
1.7.3 Viral replication
Since HuNoV cannot be easily cultured, most studies of viral replication are based on molecular studies of genome organization and viral proteins (20). Most viral replication studies derive from the observations of NV infectious clones and cell expression systems
(123, 157, 306), from pathogenesis of HuNoV in Gnotobiotic (Gn) pigs (69) and from animal CV replication studies such as PECV (65, 66, 298), FCV (137, 240) and MNV-
1(397).
31 Host target cells. The target cells for viral replication in humans seem to be the cells that
form the villi of the proximal small intestine. Results from histopathological studies from biopsies of the jejunum of adult volunteers that were infected with NV or Hawaii virus
(HV) reveal broadening and blunting of the proximal intestine villi and mononuclear cells infiltration with cytoplasmic vacuolization (8, 202). Recent evidence has been presented for HuNoV (HS66 GII/4 strain) replication in the Gn pig model (69). In this model, the virus infected enterocytes located in the villi of the duodenum and jejunum in a patchy manner. Membranous vesicles containing calicivirus-like particles were observed in the cytoplasm of HuNoV infected enterocytes by transmission electron microscopy (69).
Viral receptor. The attachment of NV rVLPs to surface epithelial cells in sections of the gastroduodenal junction and on saliva from human volunteers of secretor individuals, regardless the ABO or Lewis type, revealed that NV rVLPs use H type 1 and/or H types
3/4 as binding sites on gastroduodenal epithelial cells (248).The functional receptor of
FCV has been identified as the adhesion molecule 1. This immunoglobulin-like protein is present in tight junctions, and its expression in nonpermissive cells resulted in binding and infection by FCV strains. Blocking of this adhesion molecule with anti-feline adhesion molecule 1 antibody reduced the binding of FCV to permissive cells, resulting in strong suppression of cytopathic effects and reduced FCV progeny production in infected cells (240).
Viral replication. The FCV replication occurs in localized compartments surrounded by membranous material called replication complexes. In this membranous fraction, both full-length and subgenomic-length RNA molecules, and structural proteins (VP1 and
VP2) as well as non-structural (Pro-Pol, p30-VPg, p32, p30 and p39) proteins are
32 synthesized. In these cells VP1 protein was produced in highest concentration, and since the FCV virion contained VP1, VP2, and the VPg-linked genomic RNA, this abundance of VP1 could indicate that packaging of the genomic RNA into viral capsids could take place at the same site as RNA synthesis (137).
As described earlier, ORF1 of NoVs encodes a polyprotein that is further processed by a viral protease resulting in production of nonstructuctural proteins that are required for viral replication and RNA genome packaging (138).
The expression of ORF 3 protein in insect cells using a recombinant baculovirus expressing only the ORF3 gene resulted in several forms of the ORF3 protein varying from 23 to 35K in molecular weight. The ORF 3, 23K protein was associated with rVLPs purified from insected cells infected with a recombinant baculovirus containing the entire
3’ end of the NV genome (ORFs 2 and 3). Protein analysis of NV isolated from feces of
NV-infected volunteers contained a 35K protein as well as other higher MW proteins that were recognized by an ORF3 peptide antiserum, suggesting that this ORF3 protein is a minor structural protein of the NV virion (123). However its function is still unknown, but due to its basic characteristics it may interact with RNA and the acidic domains localized inside the virion during encapsidation of the viral RNA. Through mutagenesis studies using FCV infectious clones, it is now known that the ORF3 protein is essential for the production of infectious FCV virions (346). In FCV, the interaction between VPg protein and the cap-binding protein eIF4E and the eukaryotic initiation factor eIF4E are requirements for mRNA translation, suggesting that VPg might function as a cap structure during initiation of translation of virus mRNA (125).
33 However, the functional requirements for the components of the eIF4F complex differ among caliciviruses, since all eIF4F factors are required for FCV mRNA translation and only eIF4A is essential for mRNA translation (68).
The P41 (putative helicase) protein of South Hampton virus expressed in bacteria exhibited nucleoside triphosphate (NTP)-binding and NTP hydrolysis activities, but no helicase activity, and sequence analysis of the consensus sequences of the
SHVp41 and enterovirus protein 2C showed high similarities, suggesting that the p41 of calicivirus may have the same function as a replication complex organizer as the 2C protein of picornaviruses (306). The P48 protein forms a complex with N- ethylmaleimide-sensitive factor attachment protein receptor (SNARE) binding to the vesicle-associated membrane protein, suggesting its role in disrupting intracellular protein trafficking by possibly competing with the vesicle-associated membrane protein cellular partners for binding (100). During viral replication VP2, that is located in the 3’ end of NV mRNA, may be crucial for the regulation of the capsid protein expression and the overall viral capsid stability, because in the baculovirus expression system, when VP2 expression is blocked, the levels of VP1 and the capsid stability decrease notably (43).
Though yeast two-hybrid analysis of the binding of NV VPg to translation initiation factors the eLF3d subunit of eLF3 was identified as the binding partner of VPg. Thus this protein may have a function in initiating translation of calicivirus RNA by interacting with the translation machinery (83).
Several studies suggest that the NV proteinase is a member of the viral cysteine- proteinase family and is responsible for the proteolytic cleavage of the NV nonstructural polyprotein precursor into an NTPase (p41), a 3C-like protease (3Clpro), an RdRp and a
34 VPg, having a regulatory role in NV replication (47). Based on studies of the MD-145
ORF1 polyprotein, it is believed that during viral replication the precursor ProPol of the
polymerase is an active bifunctional enzyme retaining the proteinase and polymerase
activity. During viral replication, this protein may also interfere with the host cellular
translation by cleavage of the Poly(A)-binding protein (PABP) (219). In vitro studies
have shown that NV RNA polymerase is capable of synthesizing complementary RNA in
a primer- and poly(A)-independent manner (114). Furthermore, it has been observed that
the product of the MD-145 NV strain RdRp is twice the size of the template, suggesting
that during replication in vitro a copy-back mechanism could occur. This enzyme can
also undergo template switching, which could result in recombination between RNA
molecules during NoV replication (36).
The complete processing map and the essential cleavage sites of the FCV
nonstructural polyprotein have been deciphered. The FCV 3C-like proteinase cleaves the
N-terminal portion of the ORF1 polyprotein at cleavage sites E46/A47,E331/D332, and
E685/N686 resulting in three proteins designated p5.6,-p32-p39(NTPase)-p30-VPg-Pro-Pol.
Mutagenesis studies of FCV infectious clones showed that the cleavage between all these
gene products, except between Pro-Pol is essential for viral replication (347), and
mutagenesis of tyrosine 24 in the VPg protein is lethal for FCV suggesting that FCV VPg protein uses tyrosine for the formation of the covalent bond with genomic RNA (262).
The in vitro cultivation of PECV has provided more detailed information on the relationship between cell-signal transduction induced by IC and its effect on growth of
PEC in LLC-PK cells (111, 145), indicating that PECV replication may be dependent on an initial cAMP signaling pathway induced by IC (64). It is now known that bile acids
35 are among the active factors present in IC that are essential for in vitro growth of PEC.
Based on this information there is some speculation that since bile acids induce the proteinase kinase A signaling pathway and down-regulate IFN-mediated STAT1 activation, this would interfere with IFN-mediated antiviral innate immunity allowing
PECV to replicate in vitro and in vivo (66).
Virus release. The HuCV are released from the host’s enteric tract to the environment through the feces. The NV have also been detected in vomitus of infected patients by
IEM and RT-PCR (138).
1.7.4 Susceptibility and resistance to HuNoV infections
- Host receptors and resistance to HuNoV infections
Since the early NV challenge studies, there has always been a subset of volunteers that are resistant to NV infection and/or disease (399), suggesting that there might be genetic determinants for susceptibility and/or resistance to these agents (181).
Hemaglutination studies using blood samples from different individuals of different histo-blood group types showed that agglutination of O type red blood cells (RBCs) by
NV rVLPs occurred in all samples and that only very few samples of the B type were agglutinated by the rVLPs (136), suggesting that susceptibility and/or resistance to NV could be determined by the individuals blood group type.
The ABO histo-blood group antigens are terminal carbohydrates that are synthesized in a stepwise fashion from a precursor by different enzyme glycosyltransferases (81). Addition of a fucose in α1,2 linkage onto type 1 or 2 disaccharide precursors, by an α1,2 fucosyltransferase (FUT1 and FUT 2) produces the
36 H antigen. Inactivating mutations of these two genes lead to genetic polymorphisms at each of these two loci.
Mutations that affect the function of the FUT1 allele result in the Bombay phenotype that is characterized by the lack of ABH antigen expression on RBCs. This phenotype is rare and is found in the 10-5 to 10-6 frequency range (405). A non-functional
FUT2 gene is characterized by the absence of ABH antigens in saliva and other epithelial cell types leading to the non-secretor phenotype (409). This phenotype is found in 20% of
Europeans and North Americans. After the H antigen is formed, further addition of an N- acetylgalactosamine or galactose in α1,3 linkage, catalyzed by A and B glycosyltransferases, produces the A or B antigens, respectively. These ABH antigens are largely expressed on cells of the gut mucosa. If the alleles are inactivated by mutations, the O type is generated. If a fucose is added in α1,4 or α1,3 linkage of the N- acetylglucosamine residue of the precursor, catalyzed by the glycosyltransferase encoded by the FUT 3 gene, the Lea or Lex antigens are generated, respectively (247).
Addition of a fucose in the same position of the H type 1 and 2 antigens results in the Leb and Ley antigens, respectively (247). In the gut mucosa, the Lewis (50) antigens,
Lea or Leb are found exclusively on the surface epithelia, while Ley and H type 2 are found mainly at the glandular level (266).
In 2002, Hutson et al. demonstrated an association between ABO histo-blood group type and risk of NV infection and disease, using serum samples from volunteers that had previously participated in a NV challenge study. They showed that individuals that expressed B antigen (B and AB histo-blood group phenotype) are less likely to get infected and/or to manifest symptomatic NV disease, and that individuals of O phenotype 37 were at a higher risk of NV infection (181). This data, together with previous
hemaglutination of NV rVLPs by type O RBCs suggest that NV might bind to the
precursor H antigens on the surface of the intestinal mucosa and that the addition of the
terminal α-galactose to form the B antigen might some how alter the NV binding site on
the surface of the gut mucosa. However, other factors of resistance to NV infection and
disease might exist since some individuals of A and O phenotype do not become infected
or do not display symptoms of disease by NV (181).
Other studies also confirmed the association between blood group B and the
reduced susceptibility to symptomatic NV symptomatic infection (165). Carbohydrates
present in the gut mucosa were finally identified as a possible binding site for NV, in a
study that used NV rVLPs and various ABH type RBCs. The NV rVLPs hemagglutinated
all human type O, A, and AB, but only a few type B RBCs. The rVLPs did not
hemagglutinate any Bombay RBCs, indicating that the H histo-blood group type 2
antigens are the NV rVLP HA receptor on human type O RBCs (182).
A different study that evaluated the binding of various NoV rVLPs to saliva of
volunteers demonstrated that NV rVLPs also recognize the A, but not B antigens, in
addition to the H antigens (174). It was then determined from the saliva and synthetic
oligosaccharide assays that 8 binding patterns exist among different NoV strains and that they can be divided into two groups: the A/B and the Lewis (non-secretor) binding groups. The strains in the first group bound to types A, AB, B and O saliva of secretors but not from non-secretors, while strains in the second group bound to histo-blood group antigens of non-secretors and from type O secretors with weak or no binding to the histo- blood group types A and B secretors.
38 Thus, the current binding model suggests that strains in the A/B binding group
have a common site for the A/B epitopes and some strains may have an additional site for
the H epitopes (175).
Strains in the Lewis binding group do not bind to the A and B epitopes; moreover, these epitopes may mask the binding site to the Lewis and H epitopes (160, 161, 174,
176). This was later confirmed by transfection of blood group A or B enzyme into H- expressing cells resulting in the blockage of NV attachment to the cell surface due to H epitope masking showing that various factors such as ABO histo-blood group type, FUT
2 and FUT 3 polymorphism might have a combined effect on the susceptibility and/or resistance to NV attachment (246).
Since there is no clear distribution of NoV genogroups into the two types of two binding groups after sequence analysis of entire capsid sequences, small regions of the capsid protein might define the binding specificity of the viral capsids to the gut mucosa
(175). It has now been determined by X-ray crystallography and site-directed
mutagenesis studies of the NV capsid that the binding interface is located in the P2
subdomain of the protruding domain of the capsid protein (158, 308, 363, 364).
Cheetham et al., using Gn pigs as an animal model, also studied the influence of
histo-blood group antigens in infection and/or symptoms caused by HuNoV, and the
binding patterns of HuNoV VLPs to buccal and intestinal tissues of these animals. In that
study, it was observed that pigs expressed A and/or H or neither antigen on buccal and
intestinal tissues.
39 Forty-seven percent of the pigs that were A+ and H+ pigs shed virus compared to
non A+/non H+ pigs (25%), and more A+ or H+ pigs seroconverted compared to non-
A+/H+ pigs, although this difference was not statistically significant (70).
Oligosaccharides present in human milk may have also have a protective role
against infection and/or diarrhea by gastrointestinal viruses (377). Jiang et al., reported
that certain molecules in human milk can block binding of NoV VLPs to histo-blood
group antigens present in the saliva. In this study milk samples from secretor volunteers
but not from non-secretors blocked binding of NoV rVLPs (VA387 and NV strains) to
saliva samples. All Lewis+ Se- milk samples blocked binding to VA207 strain. Secretor
and Lewis, but not A or B antigens were detected in human milk and were responsible for
blocking NV binding to receptors, and could potentially mimic NoV receptors with a
protective role against NoV infection in breast-fed infants (190).
1.7.5 Virulence and persistence
Knowledge about HuNoV virulence is still limited, but it is known that
pathogenicity varies among the different genera of the Caliciviridae family (202). The
RHDV circulated in Britain and probably throughout the rest of Europe for at least 50
years and emerged in 1984 in China as a highly infectious disease, responsible for a high
morbidity rate among domestic European rabbits (395). It is known that the liver cell death leading to fulminant hepatic failure caused by this agent is due to apoptosis, and that programmed cell death is a constant feature in rabbits experimentally infected with
RHDV (11). Although RHDV is highly virulent for rabbits, both virulent or genetically similar avirulent RHDV are able to persist in rabbits in the presence of antibody without
40 causing severe disease and it is thought that this high stability of the RNA may play a
role in the spread and persistence of the virus in the environment (269). But, even though
eight phylogenetic groups of RHDV have been identified with significant genetic
heterogeneity in the VP60 capsid gene, the factors that are responsible for the alteration
in the phenotype of this virus have not yet been clearly identified.
Different FCV strains that vary in virulence and disease spectrum have been
described (171). The FCV are transmitted by the oral or aerosol routes and disease occurs
in the acute and chronic forms (310). Infection by the aerosol route causes a more serious
disease that affects the lower respiratory tract. The severity of the signs in the acute form
of the disease vary according to the FVC strain involved, with the more virulent strains
causing fever, pneumonia and ulcers of the tongue and nostrils. Infection by the less
virulent strains usually does not reach the lungs (310).
In 1998, a new strain of FCV suddenly appeared among cats of a veterinary
practice and caused a severe systemic hemorrhagic-like fever with high mortality rates
(179). The virulent systemic feline calicivirus, was weakly neutralized by an antiserum
against the universal FCV-F9 strain, and subsequent sequence analysis of the capsid gene
and the whole genome revealed that not all virulent systemic feline calicivirus group
together and that these mutant strains have emerged from different lineages of FCV.
Various mutations were detected in the same region (from nt 398-592) of the virulent
systemic feline calicivirus capsid forming an extra predicted glycosylation site in the
amino acids in the region (113). It is speculated that this change in the capsid protein
could be responsible for new host-virus interactions changing the tropism and infectivity of this agent.
41 1.7.6 Apoptosis
Several morphological and biochemical changes take place during apoptosis. The main features are cell shrinkage, nuclear chromatin condensation and proteolysis of important cellular proteins by the cysteine proteases, caspases. There are two main pathways by which apoptosis can occur: the extrinsic (death receptor) and the intrinsic
(mitochondrial) pathways (49, 129). In the extrinsic pathway, members of the tumor necrosis factor (TNF) superfamily that have death domains (death receptor), such as Fas, bind to their ligand (Fas ligand) and upon binding, their intracellular domain associates with an adaptor protein, the Fas-asociated death domain, directly or indirectly via the tumor-necrosis factor receptor-associated death domain. The Fas-asociated death domain also interacts with pro-caspase-8 to form a complex at the receptor called the death inducing signalling complex. Once assembled, the death inducing signalling complex induces the activation of caspase-8, which in turn precipitates the activation of downstream effector caspases such as caspase-3 (370). In the mitochondrial pathway, the cytochrome C interacts with the apoptosome via the apoptotic protease-activating factor
I, and the recruitment of pro-caspase-9 into the apoptosome leads to the activation of caspase-9, that mediates the activation of other caspases such as caspase-3 and –7 (49). In both pathways the proteolysis of important cellular enzymes results ultimately, in cell death.
As described above, apoptosis is involved in the pathogenesis of the fulminant hepatic failure induced by RHDV infection, the apoptotic process affected mainly hepatocytes, but macrophages and endothelial cells were also stained by viral antigen, by immunohistochemistry, and exhibited signals characteristics of apoptosis (11).
42 Apoptosis has also been described in cultured cells infected with the FCV (314,
348). Infected cells showed chromatin condensation, DNA fragmentation and caspase
activation, and only recently, the molecular mechanism of apoptosis in FCV-infected cells was defined. At 6 hrs post-infection, the FCV F9 induced translocation of phosphatidylserine to the cell outer membrane and release of cytochrome c from mitochondria in Crandell-Rees feline kidney cells. The release of cytochrome c from the mitochondria triggered the activation of caspase-9 that mediated the activation of caspase-3 (275). The authors speculate that, since FCV protease shares common features with the picornavirus superfamily 3C proteases that can trigger apoptosis via the mitochondrial pathway, the p39 FCV protein which contains the NTP-binding domain could be responsible for triggering apoptosis in FCV-infected cells.
Signs of apoptosis were also detected in Gn pigs infected with GII.4 HuNoV. In the intestine of the pigs, infected cells were located predominantly at the tips or sides of each villus, and viral capsid antigen could be detected at various locations within the cell cytoplasm, but never in the nucleus. In infected cells, the intracellular organization was disrupted showing nuclear displacement and decreased number of organelles, with the presence of vesicles in the cytoplasm filled with calicivirus-like particles. Apoptotic and necrotic cells were also detected in increased numbers in the HuNoV-infected pigs by the
TdT-mediated dUTP nick end labeling assay (69).
The HuNoV-associated apoptosis has also been documented among intestinal transplant patients. In those patients, HuNoV infections caused similar apoptotic features to those seen during allograft rejection, such as increased apoptosis of crypt enterocytes leading to confusion during the differential diagnosis between HuNoV- and acute cellular
43 rejection-induced apoptosis (208). However, some specific features of HuNoV-induced
enteritis such as disarray of surface epithelial cells and increased apoptosis of the surface
epithelium and superficial lamina propria were later identified, aiding in the differential
diagnosis from acute cellular rejection (268).
1.8 Epidemiology
Acute gastroenteritis is a major public health concern throughout the world. In
developing countries, it is responsible for more than 25% of all deaths among children
under 5 years of age (53, 386). In developed countries diarrheal diseases are also
responsible for causing a large disease burden associated with high childhood morbidity and hospitalizations (53, 386). The HuNoVs constitute the major cause of nonbacterial, epidemic gastroenteritis in the world, and in the US alone they are responsible for 23 million cases of gastroenteritis and 50,000 hospitalizations every year (260). These agents are distributed worldwide and sporadic outbreaks, usually associated with food and water contamination, have been detected in samples from people of all ages (1, 75).
1.8.1 Molecular epidemiology
Due to the great variability and to the fastidious character of the HuNoVs, the detection and characterization of strains involved in numerous outbreaks (3, 5, 6, 54) have relied mainly on the ongoing development of molecular detection methods (20).
However, the proportion of NoV foodborne outbreaks vary greatly from country to country, and it may be underestimated in parts of the globe where there are no well- established surveillance systems and the detection methods are still precarious (54).
44 A balance between the use of broadly reactive and highly sensitive primers (131,
201) followed by sequence analysis, mainly of the capsid and polymerase regions, of
strains from different outbreaks in different parts of the world has provided the
classification of NoV strains in genotypes or genetic clusters within the different
genogroups (15). It is now known that many different genotypes of HuNoVs circulate at
the same time in the population causing sporadic cases and outbreaks (102). Different
strains detected from a single outbreak are usually very similar. Therefore, when samples
from different patients have identical strain sequences, it is likely that they originated
from a common source of infection (121, 152), except in the cases of waterborne
outbreaks due to sewage water contamination where more than one source may be
involved, and consequently different strains can be detected (127).
In the US, the RT-PCR detection of HuCV has been adopted at the “Centers for disease control” (CDC) for the routine detection of NoVs since the early 1990s (263).
From 1997 to 2000, the CDC examined data from more than 8,000 different foodborne outbreaks that had been previously reported and also new data from 6 other states. In
1991, only 1% of the outbreaks were attributed to HuNoV; however, this number increased to 12% in 2000, and from 1998 to 2000, only 11 states were responsible for reporting 76% of NoV outbreaks. In 2000, 50% of foodborne outbreaks detected in 6 states (Georgia, Minnesota, Ohio, Florida, Maryland and New York) were NoV-related.
Samples from another 226 outbreaks that occurred between July 2000 and June 2004, were sent to CDC for CV testing. Eighty-one percent of these samples were positive for
HuCV by RT-PCR and nucleotide sequencing. The most common settings where these agents were found were nursing homes, retirement centers and hospitals, with the person-
45 to-person contact being the most common transmission mode. The GII HuNoV were the most often detected strains (79%) followed by GI strains (19%); however, SaVs were found in only 2% of the samples (48).
In Germany, 316 stool samples originating from over 150 nonbacterial gastroenteritis outbreaks during the years 2001-2004 were analyzed for the presence of
NoV by RT-PCR using primer pairs targeting the RdRp gene of both genogroups I and II.
Forty-three percent of the outbreaks occurred in nursing homes and elderly care centers, and 24% in hospitals. The RT-PCR followed by sequencing showed that 12 genotypes were involved in the outbreaks, with co-circulation of GI and GII detected in the same place. However, GII NoV alone were responsible for 95% of the outbreaks, with GII.4
(Grimsby-like strain) being the most prevalent strain during 2002-2003 (183). The data suggest that routinely application of molecular assays will provide a more accurate estimate of NoV-related outbreaks and their most frequent settings and contamination sources, providing important data for the control of Nov-induced illnesses around the world (393).
1.8.2 Seroprevalence
The use of recombinant baculovirus for the development of rVLPs of different
HuCV strains (28, 35) has had a great impact in the investigation of HuNoVs seroprevalence in the general population worldwide, shedding light on the duration of antibody responses after natural infection, and subsequent re-exposure of some individuals to these agents (30, 284).
46 The prevalence of antibodies to NV was determined in more than 3,000 serum samples collected between January 1991-May 1992 from patients from hospitals, outpatient clinics and from blood donors in various regions of England. The NV VLPs were used as coating antigens for detection of the IgG antibodies in the serum samples, and the patients’ ages varied from <6 months to > 90 years. Seventy-three percent of all the samples were positive for NV antibody. A high seroprevalence (75%) was found in <
6-month-old infants, which could reflect the presence of maternal antibodies. Seventy- percent of school-age (5-9-years-old) children had IgG antibodies against NV. This prevalence increased slightly for the young adults from 20 to 29 years of age (81%), with the prevalence among the elderly (>90 years of age) remaining elevated (87%). When the samples were divided according to the geographic area of collection, it was observed that antibodies against NV were acquired earlier in life in the urban areas, where 30% of the
6-11-month-old children had antibodies compared to none of the children in the more rural areas. However, in the adult population (≥ 30 years old) no differences in seroprevalence according to regionally were found (128).
In the US, the antibody responses to NV and Mexico (MX) strains rVLPs were measured in the acute and convalescent serum samples of individuals involved in outbreaks that were associated with MX-like strains and in outbreaks associated with other GII HuNoV. Most outbreaks occurred in hospitals, and in residential homes for the elderly, and the individuals enrolled in the study included patients and staff. The IgM responses specific to MX were observed in only 18% of the acute phase sera and 49% of the individuals had at least a four-fold increase in IgG titers to MX VLPs with 10% of them having anamnestic responses to NV VLPs in the convalescent serum samples (151).
47 In the Netherlands, homologous and heterologous antibody responses in the sera of adults
that were involved in 13 outbreaks caused by 4 different HuNoV genotypes: LV (GII.4),
MX (GII.3), HV (GII.1) and Leeds virus (50) (GII.7) (50) were also characterized. The age of the individuals that participated in this study varied from 19 to 100 years. A four- fold increase in antibody titer was detected in 62% of all individuals. Seroconversion for
IgA and IgG antibodies was observed for all genotypes after LV infection but with higher percentage of positive samples for the homologous response. Seroconversion to MX virus was also maily homologous, but responses to LV were also present. Of interest, one individual seroconverted with IgA after HV infection but not with IgG, and no IgG homologous seroconversion responses were observed after LE infection; however, heterologous IgG responses were detected for LV, and MX (20% and 40%, respectively)
(315). In Italy, 30% of the samples collected from outpatient clinics and hospital patients, with an age range from 1 day to 95 years old, had antibodies against South Hampton virus (SHV), another GI HuNoV (300).
In Japan and Southeast Asia, a low prevalence (6-22%) of antibodies to NV was detected in children from 4-months to 6-years of age. From school age until adulthood the prevalence of antibodies increased steadily until it reached 98% in the adults in the ≥
50 year-old age group. These findings showed that NV infections seem to be rare in infants but are very common among adults in Japan and Southeast Asia (284).
In Beijing, China, 89% of the samples collected from individuals of different ages during April, 1996 and March, 1997 had IgG antibodies against NV and 91% against
MX. Infants had a high seroprevalence (99% for NV and 94% for MXV) at birth; this percentage decreased from 7-11 months of age (41% for NV and 36% for MXV) and a
48 sharp incrase was detected during early childhood reaching 100% for NV and 98% for
MXV at 8-9 years of age, suggesting that infection by NV and MxV are common in this population (197).
In Kenya, the acquisition of serum antibodies (IgG) to HuCV (NV, MX and SaV) occurred early in childhood at about 1-2 years of age, with a higher prevalence of antibody (80-90%) to MX and SaV compared to NV (60%) (274). In Kuwait, NoV infections are also acquired early in age, with 95% of infants from 4-11 months of age having antibodies to MX (88). In Brazil, the seroprevalence of IgG antibodies to NV among children living in an urban shantytown in the Northeast of the country was evaluated. Thirty-six percent of infants from 4-12 months had antibodies to NV; at 2 years of age the seroprevalence increased to 70% reaching 80% by 4 years of age (362).
The data from all these studies show that in developed countries, infections by
NoV are acquired gradually: by age 50 at least 50% of individuals have antibodies. In comparison, in some developing countries, the NV seroprevalence in young children varies from 80%-100% (45, 88).
1.8.3 Incidence
For a period of 2 years, three hospitals in the US collected fecal samples from children up to 5 years of age with acute gastroenteritis. Overall, 156/1840 (8.5%) of the samples were CV-positve. After sequencing, 7.1% of the samples were characterized as
NoV and 1.4% as SaV by RT-PCR using the primer pair 289/290 that targets a conserved region of the CV RdRp gene. The phylogenetic analysis of the NoV strains showed that within GI and II, 98% of the NoV strains were grouped into genetic clusters with no
49 known prototype in GenBank. The analysis of 24 SaV strains showed that 50% of them grouped with the London/92 strain in one genogroup and the rest in three other proposed genogroups. The data showed that both NoV and SaV are common gastroenteritis agents in young children and that there is a high diversity among the circulating strains (412).
In a prospective cohort study done in the Netherlands, the natural history of
HuCV infection was investigated in over 4000 subjects with ages ranging from 0-≥65 years for a period of 6 months. Both NoV and SaV were detected in fecal samples by RT-
PCR. Infection by NoV was common in all ages, but SaV infection was detected mainly in children < 5 years of age (316). In England, NoV were detected in 13% of the fecal samples from children under 5 years of age, by RT-PCR, and SaV were detected in only
1% of the samples (334). In France in a study of over 400 cases of gastroenteritis in children between 1995 and 1998, HuCV (NoV and SaV) were detected in 14% of the fecal samples by RT-PCR (51).
In Melbourne, Australia, a community-based gastroenteritis survey was conducted. A subset of 638 gastroenteritis fecal samples from 456 individuals that had had at least one episode of gastroenteritis during a 2 year period was analysed for the presence of bacteria, viruses and parasites. The NoVs were detected in 11.4% of all the
638 fecal samples tested, by RT-PCR, both in adults and in children. Two percent were identified as GI and 10% as GII HuNoVs (249). Another study in the United Kingdom found NoVs in 7% of gastroenteritis cases in a community-based study (373). Farkas et al. reported that up to 19% of cases of acute gastroenteritis in Mexican children with less than 2 years of age are caused by NoVs (103).
50 In most countries, including the US, most of the information on HuCV incidence
comes from outbreak-related incidents, and will be discussed in detail in the “viral
outbreaks” section below.
1.8.4 Viral outbreaks
Outbreaks caused by HuNoV involve persons from all age groups, may occur
during any season and in various locations (48). The most common sources of outbreaks
are contaminated food and water, with secondary person-to-person transmission being
common. They may occur in many different settings such as: hospitals, nursing homes,
schools, and swimming pools. The outbreaks generally last for about 1 week, with longer
outbreaks occurring when new susceptible individuals are in contact with contaminated
ones, generally in a setting where there is a persistent source of infection. During the
outbreaks, vomiting is the predominant symptom among children and diarrhea is
common among adults (206).
According to CDC, from 1976 to 1980, 42% of 74 outbreaks of acute nonbacterial
gastroenteritis were attributed to NV (206). However, since then most of the outbreak identification was based on fourfold increases in NV antibody titers from serum pairs
from outbreak cases and on IEM observation of fecal samples from individuals involved
in the outbreaks. However, many of those cases that were attributed to NV could had been caused by other GI NoV or even by NoVs belonging to other genogroups.
With the development of RT-PCR primers targeting RdRp regions of both GI and
GII, new probes and the use of sequence analysis to detect and characterize viral RNA in fecal samples and with the development of rVLPs from different viral strains to use as
51 antigens in immunoassays for detection of antibodies and seroconversion, the
identification of the viral strains causing the outbreaks has become more efficient and
accurate.
Between August, 1993 and July, 1997, 152 outbreaks of nonbacterial acute
gastroenteritis were investigated by the CDC. The settings included health care facilities,
restaurants, institutions such as schools and prisons, and cruise ships. All fecal samples
were characterized by RT-PCR using primers targeting a 123bp region of the RdRp gene; in addition a 322-bp region of the capsid of at least 1 specimen from each genogroup II outbreak was sequenced. The results showed that until the beginning of 1995, many strains were involved in the oubreaks and not a single strain could be related to 2 different outbreaks in the country. However in April, 1995, the characterized strains from many different outbreaks had almost identical sequences, and those strains belonging to
GII.4 NoV were defined as the “95-96-US” subset, and their circulation predominated until 1996. Viruses with characteristics of this same strain were simultaneously detected in 7 other countries on 5 continents (282). In North Carolina, the investigation of 16 gastroenteritis outbreaks from 1995-2000, revealed that the outbreak strains belonged to the various genetic clusters: GII.4, GI.3, GI.4, GII.2, GII.5, GII.7 and GII.13. The five
GII.4 strains circulating during the 5-year period were identified as the: “95/96 US common strain”, and most of the outbreaks in North Carolina (86%) were foodborne
(376). During the 2002 year, the CDC received fecal samples from 48 different acute gastroenteritis outbreaks in the US.
52 The common settings included restaurants, catered events, cruise ships, school,
child-care centers, assisted-living facilities, and sporting events. Seventy-three percent of
the samples were NoV-positive and 41% of those were associated with the same GII.4
strain named “Farmington Hills” (4).
During 2002 and 2003, an increase in NoV outbreaks was reported in Oxfordshire
(United Kingdom) hospitals and variants related to the GII.4 strain (Lordsdale virus-like) were identified as the cause of the outbreaks. A high rate of mutation was detected among these variants and the determination of longer sequences from samples from each outbreak allowed the connection or separation between outbreaks to be made (89).
During the same period, other European countries such as the Netherlands, Germany and
Finland also reported an increase in NoV activity caused by a new variant of the GII.4
strain that had a consistent mutation in the polymerase gene (231). According to the
Foodborne Viruses in Europe Network, nine countries in Europe reported an increase in
NoV activity during October and November of 2006, compared to the same period in
2004 and 2005 with the majority of the outbreaks being caused by GII.4 variants (216).
1.8.4.1 Sources of NoV outbreaks
Foodborne NoV outbreaks account for a great majority of HuCV-related outbreaks in the world. During 1981-1998, 41% of all confirmed foodborne outbreaks in
Minnesota were NoV-related (86). Oysters have been identified as an important NoV contamination source. Oyster samples from 11 countries in Europe, America, Asia and
Australia were analyzed for the presence of HuCV, and RNA from GI and GII NoVs were detected in oyster samples from six out of the 13 outbreaks (73). Human and animal
53 CV were also detected in oysters from different coastal regions of the US. Twenty-
percent of the samples were positive for GII HuNoV, and animal enteric calicivirus
(ECV) were detected in 22% of the samples (77), confirming the importance of raw
oysters as potential causal agents for NoV gastroenteritis.
During 2006, NoV contaminated raspberries were identified as the cause of 4
different outbreaks in Sweden. All the suspected raspberries had the same brand and
originated from the same distributor in Sweden who imported them from China (169).
Berries have been the cause of several outbreaks in many European countries in recent
years. Since 2000 Sweden has had 11 outbreaks of gastroenteritis caused by NoV and in
all of them, the suspected food contained raspberries (169).
Sewage water should also be considered as an important potential source for
HuNoV outbreaks (14). A high number of HuNoV particles are shed during
gastroenteritis episodes and, consequently, a high virus load is dispensed into the sewage
system, that is then treated with a chlorine concentration that is not sufficient to inactivate
NV, constituting a potential risk for outbreaks in the human population. (210).
It has been estimated that the average NoV concentrations in raw sewage are approximately 105 pdu per liter compared with 103 pdu/L of treated sewage water (378).
This treated water is then used as primary material for drinking water production, used in
pools and other recreational activities. During a study that sampled sewage water during a one year period, the most prevalent variant detected in both raw and treated sewage water was genogroup/genotype (GG) IIb, together with GGI.6 Sindlesham and GGII.2
Melksham (378). It is also estimated that one vomiting incident can contain more than 30 million NoV particles that could potentially contaminate the immediate environment and
54 surfaces with viral-rich aerosols (61). The challenges in detecting enteric viruses in environmental water include the presence of PCR inhibitors and the need for concentration of a large volume of water in order to detect small numbers of viral particles (261).
A GII multistrain NoV outbreak among snowmobilers was caused by NoV contaminated well-water in Wyoming in 2001(14). In 2004, another waterborne outbreak of gastroenteritis with multiple etiologies was detected among visitors and residents of the South Bass Island, a resort island on Lake Eire, Ohio. Combined epidemiological and environmental investigation linked sewage-contaminated ground water to the possible source of the outbreak. However many agents such as Escherichia coli, Campylobacter jejuni, Salmonella and NoV could have caused the gastroenteritis since multiple fecal microbes were identified in the fecal samples from the individuals involved and in the water tested (291).
Other settings such as nursing homes, schools, and cruise-ships have been identified as potentially important NoV outbreaks locations. Fecal samples from
Maryland nursing home residents, who became ill during 20 outbreaks of gastroenteritis in 20 different nursing homes from November, 1987 through February, 1988, were collected and tested by RT-PCR for HuCV. Fifty-two percent of the samples were NoV- positive, and sequencing data identified six distinct genetic clusters within NoV GI and
II, with GII.4 variants (MD-145) being the predominant strain (134).
In nursing homes near Tel-Aviv, Israel, a high case-fatality rate of NoV gastroenteritis that affected both the elderly residents and staff occurred during 2002.
Sequencing data from all the samples positive for NoV revealed that they all belonged to
55 the same GII genetic cluster and were 90% identical. The closest NoV strain by blast
analysis was the Hu/NVL/UKB7s2. Since most of the residents of the nursing homes
were bedridden, the spread of the infection was by direct contact from staff members to
residents (60). The CDC recommended that all staff members should vigorously wash
their hands with water and soap for more than 10 seconds, discard gloves and aprons after
treating each patient, isolate the affected from the unaffected use disposable plates and
cutlery during outbreaks as measures to stop the person-to-person spread of the disease
among the elderly home residents (295).
The closed environments of cruise-ships, with consecutive cruises with new
passengers coming onboard in a relatively short period of time constitute risk settings for
NoV outbreaks (2). A large NoV-related outbreak affected 6 consecutive cruises on the same ship, although thorough disinfection of the ship was performed after the second cruise. During the investigation, epidemiologic analysis revealed that the initial source of the gastroenteritis outbreak was foodborne, with subsequent person-to-person spread of the viruses. Identical sequences of the same strain were detected in stool samples from sick passengers before and 1 week after the ship’s cleaning, suggesting persistence of the strains with subsequent infection of new susceptible individuals (186).
Another outbreak of both astrovirus and NoV on a research ship surveying Tokyo
Bay caused acute gastroenteritis in 26 of 37 crew members. The NoVs were detected by
RT-PCR in 49% of the fecal samples from the affected crew members and astrovirus were observed in 38% of the cases. Mixed infections of both NoV and astrovirus were identified in 6 cases. Most NoV-positive samples belonged to GII. One NoV isolate
(Minato.14) was identified as a possible recombinant between a NoV strain from GII.6
56 and a GII.1 strain (328). In this type of setting, infected cruise members could also function as reservoir of infection between cruises, and newly infected cruise members can also go on land and cause new outbreaks in different settings.
1.8.5 Interspecies transmission and zoonosis
It is now believed that marine mammals were the actual natural hosts of members of the Vesivirus genus and that since the early 1930’s the increased feeding of raw fish and sea lion carcasses to pigs started a rapid and uncontrolled pig to-pig spread of VESV, that reached the swine population of many US states (342). Other animals such as mink when fed calicivirus-infected raw seal meat also became infected with VESV virus (329), and a HuCV isolate of the SMSV, designated SMSV-5 Homosapien-1, was recovered from vesicular lesions of a laboratory worker, who had been recently working with the SMSV-5 strain in the lab. Natural hosts for the SMSV-5 include seals, cattle, whales, donkeys and fox, and susceptible hosts include opaleye fish, horses, domestic swine and primates. Feline calicivirus causes disease not only in dogs but also in seals and the strain FCV-F9 affects domestic cats and cheetahs and dogs are also susceptible
(342). Taken together the data show that the interspecies movement of at least, some members of the Caliciviridae family is a reality.
More recently, the finding of NoV sequences in stool samples from pigs
(358, 384), the discovery of bovine and swine NoV that are closely related to HuNoVs
(226, 384), the detection of recombinant human (189), bovine (289) and swine NoVs
(384) raises concerns that animals may constitute a reservoir for HuNoV.
57 The presence of serum antibodies against HuNoV in animals and of antibodies to animal NoVs in humans has also recently been evaluated. A seroprevalence study of NoV in swine revealed that 63% of pig serum samples from the US were positive for antibodies against NV (GI) and 52% for HV (GII) (104). Serum antibodies to bovine
NoV were also present in veterinarians and the general population in the Netherlands.
The IgG antibodies to recombinant bovine NoV capsid antigen (rBoNoV) were more commonly detected among veterinarians (28%) than in controls (20%). Although some cross-reactivity of IgA and IgG antibodies to rBoNoV and to rNV was detected, 26% of all samples positive for rBoNoV antibodies showed high reactivity to rBoNoV but low reactivity to rNV, suggesting a specific response to the bovine antigen (392). An investigation of vesivirus seroprevalence and viremia among blood donors reveled that the seroprevalence to VSV antigens among different groups of blood donors varied from
12-47%. Moreover, VSV RNA sequences were detected in 10% of the sera tested by RT-
PCR (338). These data provide support for the public health concerns for the zoonotic potential of these fastidious viruses.
1.9 Immunity
1.9.1 T cells
In humans, naïve CD4+ T cells differentiate into two subsets of functional T helper cells upon activation: T helper 1 (Th1) and T helper 2 (Th2) cells. They all express an αβ T cell receptor but differ in function. The key determinants for the Th1 or Th2 outcome are IL-12 and IL-4 influence through STAT4 and STAT6 signaling, respectively.
58 Together with antigen dose, co-stimulatory molecules and certain transcription
factors such as T-box transcription factor (T-bet) for Th1 development and the zinc-
finger transcription factor (GATA3) for Th2 characteristics (272).
-T helper 1 cells (Th1). This subset of CD4+ T cells produce type 1 cytokines (IL-2,
IFN-γ and others) that support macrophage activation, generation of cytotoxic T cells and
the production of opsonizing antibodies (272). The maturation of naïve CD4+ T cells into
mature Th1 effector cells and the production of IFN-γ is stimulated by secretion of the pro-Th1 cytokine IL-12 (178).
-T helper 2 cells (Th2). This subset of CD4+ T cells produce type 2 cytokines (IL-4, IL-
5, IL-13, and others) that support B-cell activation, the production of non-opsonizing
antibodies, allergic reactions and the control of extracellular parasites (272).
-T regulatory cells (T-reg). Two subsets of T-reg cells have been described in humans.
They are both generated through CD3 and CD46 activation and have two distinct
mechanisms for effector T-cell suppression: secretion of IL-10 and granzyme synthesis.
-Natural CD4+CD25+ T- reg cells: these cells originate in the thymus, have self-antigen
specificity and act mainly through a contact-dependent mechanism that probably involves
granzyme A in humans. They express the transcription factor forkhead box P3 (FOXP3)
that seems to be a crucial factor in the induction of the T-reg population, and the IL-2
receptor α-chain (CD25). These cells require exogenous IL-2 for their function and
expansion (209).
59 -Inducible or adaptive T-reg 1 (Tr1) and Th3 cells. These cells are generated in the periphery against both self and foreign antigens. They also require IL-2 as a growth factor and mediate their suppressive effect through secretion of IL-10 (Tr1 cells) or transforming growth factor-β (Th3 cells). They might or might not express FOXP3 and have variable expression of CD25 (209).
-CD8+ cytotoxic T lymphocytes. These T cells are characterized by the secretion of
certain cytokines such as IL-2 and IFN-γ during antigen clearance but can also lyse virus-
infected cells and tumor cells through expression of cytotoxins such as perforin and
granulysin (156). The IFN-γ production by these cells is greatly influenced by the pro-
Th1 cytokine IL-12 (178).
-Memory T cells. During infection, naïve T cells recognize an antigen, in the context of
the major histocompatibility complex, for which their TCR have high affinity and under
the influence of co-stimulatory molecules present on the surface of antigen-presenting
cells (APCs) they proliferate and differentiate into effector cells. After the infection is
cleared, most of these cells die by apoptosis; however, some of these antigen-specific
cells survive in a lower state of activation constituting a pool of memory T cells.
Compared to naïve T cells, memory T cells express higher levels of β1 amd β2 integrins
and other surface molecules such as IL-2R β-chain that may play a role in the
maintenance of memory T cells and the common leukocyte antigen (CD45) that in
memory T cells is expressed in the lower molecular weight form. Memory T cells also
differ functionally from naïve T cells. They have less stringent requirements for
activation than naïve T cells, are able to respond to lower concentrations of antigens and
are less dependent on costimulatory signals than naïve T cells. They proliferate faster and
60 display effector functions sooner after activation and also seem to have an increased capacity to interact with APCs compared to naïve T cells. The survival of memory T cells is antigen-independent and it is different from naïve T cells, that do not depend on interactions with MHC for survival (38).
1.9.2 B cells
The B cell production in adult vertebrates initiates in the bone marrow, and there development occurs through several stages that represent a change in the genome content of the antibody loci. The antibody is formed by two light and two heavy chains, and in the H loci there are three regions named variable, diversity and joining. These regions randomly recombine, by the action of lymphoid-specific recombinase activating genes
Rag 1 and Rag 2 together with the terminal deoxynucleotidyl transferase, producing a unique variable domain in the immunoglobulin of each individual B cell. Similar rearrangements occur for light chain locus except that there are only two regions, namely variable and joining. When the B cell fails in any step of the maturation process, it will die by apoptosis. If it recognizes a self-antigen during the maturation process, the B cell will become suppressed (anergy state) or undergo apoptosis. B cells are continuously produced in the bone marrow, but only a small portion of newly made B cells survive to participate in the long-lived peripheral B cell pool. After antigen activation, mature naïve
B cells (located in the B-cell follicles in secondary lymphoid organs) migrate to the edge of the follicles where they are stimulated by T cells. If they express the appropriate surface molecules such as the B-cell lymphoma 6, inducible T-cell co-stimulator ligand,
61 CD40 and B-lymphocyte-induced maturation protein 1 this interaction with T cells results in the formation of short-lived plasma cells and the establishment of germinal centers in the follicles of secondary lymphoid organs. These germinal centers are populated by B cells, T cells, macrophages, and specialized germinal-center dendritic cells, in addition to the follicular dendritic cells that have stromal origin. The germinal centers in the lymphoid tissues have a dual staining pattern due to the difference in cellular composition at either pole or zone. The dark-staining pole (the dark zone) consists mainly of lymphocytes and the light-staining pole (the light zone) contains numerous cytoplasm-rich follicular dendritic cells (402). The proliferating antigen- specific B cells are located in the dark zone wherease the non-proliferating immunoglobulin-expressing B cells are in the light zone. The latter can differentiate into memory B cells or plasma cells, or die by apoptosis if they are not stimulated by their cognate antigen (258).
- Memory B cells. These cells recirculate in the periphery and long-lived plasma cells generally accumulate in the bone marrow (258). They are specific to the antigen encountered during the primary immune response, are able to live for a long time, and can respond quickly following a second exposure to the same antigen, whereas plasma cells produce and secrete large amounts of antibodies, that bind to pathogens, facilitating their phagocytosis by macrophages and other phagocytes. In mice, T-cell dependent memory B cells express class-switched antibody, CD38 and B220, they have the ability to bind small amounts of the lectin peanut agglutinin, they have somatically mutated V gene segments and they costitutively express the co-stimulatory molecules CD80 and
62 CD86. Most human memory B cells are similar to those of mice, but they also express
CD27 that is a marker for the identification of human IgM+ memory B cells that have
somatically mutated V gene segments. Memory B cells are generated in response to both
T cell-dependent (maily protein antigens) and T cell-independent antigens
(carbohydrates) (366).
1.9.3 Dendritic cells
Dendritic cells (DC) are antigen-presenting cells that have the ability to not
only participating in the front line defense mechanisms of the immune response or the
innate immune responses, but also in inducing and regulating antigen-specific immune
responses. Myeloid or lymphoid DC progenitors mature into circulating precursors upon
IL-3 exposure, and then home to tissues where they reside as immature cells with high phagocytic capacity. Upon antigen exposure they capture the antigen and migrate to the
effector sites where they further differentiate into DC subpopulations that have specific
functions under the influence of chemotactic, differentiation factors and pathogens
products such as LPS or nucleic acid. In the lymphoid organs some of these DC can
initiate immune responses by processing and presenting the antigen to CD4+ T helper
cells. These helper T cells aid the DC in their terminal maturation that, in turn, induces
lymphocyte expansion and differentiation. The DC also activate B cells by cell-to-cell
contact allowing the B cells to migrate into different areas where they mature into plasma
cells that produce and secrete antibodies functional in antigen neutralization (27).
63 Studies of mice revealed that the lymphoid and myeloid DC subsets differ in
phenotype, localization and function. They both express high levels of CD11c, MHC
class II molecules, and the co-stimulatory molecules CD86 and CD40 (244, 398). The
marker that can distinguish these two subsets is CD8α that is expressed as a homodimer
on the lymphoid DC, but is not present in the myeloid subset (398). The lymphoid DCs
secrete higher levels of IL-12 and are less phagocytic than myeloid DCs. The IL-12
induces production of IFN-γ in lymphoid but not in myeloid DCs (286).
In humans, at least two subsets of DC precursors circulate in the blood: CD14+
CD11c+ monocytes and lineage markers (CD11c, CD13 and CD33)-negative (LINneg)
CD11- IL-3Rα+ precursor DC. The LIN neg CD11c+ cells may represent a third precursor.
The CD11c+ CD14 monocytes, as well as LIN neg CD11C+ blood DCs develop into
immature DCs under the influence of granulocyte/macrophage colony-stimulating factor
(GM-CSF) and IL-4 or TNF. The CD11c+ blood DCs can differentiate into Langerhans
cells in the presence of TGF-β. The survival of CD11c- DC precursors (plasmacytoid
monocytes) is dependent on IL-3 and their maturation on CD40-L. The monocytes DC
precursors express significant GM-CSFRα and low IL-13Rα, whereas CD11c- DC
precursors display low GM-CSFRα and high IL-13Rα. The immature monocyte-derived
DCs have high endocytic/phagocytic capacity while the immature CD11c- DC do not.
Plasmacytoid monocytes isolated form human blood also express CD4, CD36,
CD45RA, CD62L (L-selectin), CD68, CXCR3, immunoglobulin-like transcript receptor
3 (ILT3), and the cutaneous lymphocyte-associated antigen. Since these cells express the
L-selectin that binds the mucosal addressin-cell adhesion molecule-1 (MAdCAM-1), the
CXCR3 that binds the gamma interferon-inducible protein-10 and also the monokine 64 induced by interferon gamma chemokines, and because they were also found in the
lumen and in close proximity to the high endothelial venules (HEVs) in inflamed lymph
nodes, it is suggested that these cells could enter the lymph nodes from the blood stream
at HEV during inflamation. After being infected with influenza virus and after being stimulated with CD40L-transfected cells, these ILT3+/1- cells produced large amounts of
IFN-α (62).
In the mouse, subsets of the pDCs that express different cell markers are found in the sub-epithelial dome, in the interfolicullar regions of the Peyer’s patches, and in the T zones of the mesenteric lymph nodes and it is speculated that they can also be found in the lamina propria (198).
1.9.4 Cytokines
Cytokines are glycoprotein hormones that can be produced in all tissues and by most cells and participate in tissue defense, growth and repair processes. The induction of cytokines in a cascade fashion upon tissue damage or inflammation is crucial for the response of the organism to pathogens and also very important for maintaining tissue homeostasis. Cytokines also act on the regulation of both the innate and adaptive immunity at the level of antigen presentation, clonal expansion, cellular differentiation and regulation of effector function (172).
-IL-1. IL-1 is a major pro-inflammatory cytokine that acts in the regulation of the immune system. The IL-1 family is composed of IL-1α, IL-1β, the inhibitory IL-1 antagonist (IL-1 Ra) and IL-1γ (IL-18). They bind to receptors that have folds in the extracellular portion and Toll/IL-1R (TIR) motif in the intracytoplasmic portion. The IL- 65 1β in combination with CD40L induces monocyte-derived DC to secrete high levels of
IL-12, triggering the production of high levels of IL-6 and IL-1 as well (390).
-TNF. The TNF cytokine family includes the lymphotoxin (LT)α, LTβ, and LIGHT.
These cytokines have a crucial role as mediators of the organogenesis of the peripheral
lymphatic organ system. The TNF is a pro-inflammatory cytokine that is produced by
cells of the innate immune system, including monocytes/macrophages, natural killer
(NK) cells, mast cells, and neutrophils (305).
The IL-12 family members include IL-6, IL-12, IL-23 and IL-27, and all of them
are produced in response to microbial and host immune stimuli, such as Toll-like
receptors and interferons. They are produced by many cells such as monocytes,
neutrophils, and B cells.
-IL-6. The IL-6 is a pro-inflammatory cytokine that is associated with the early immune
response to infection and with the regulation of innate and adaptive immunity. The IL-6
also stimulates IgA B cell development in vitro (63), and in synergy with IL-5 it
promotes higher levels of IgA secretion by IgA-commited cells (217).
-IL-12. The IL-12 is a key cytokine in the innate immune system, produced mainly by
macrophages and DC, and it was initially associated with the innate recognition of
pathogens but is also produced in a T-cell dependent manner through the engagement of
APCs with CD40L on T cells during the development of adaptive immune responses. It is
considered a pro-Th1 cytokine that has a pivotal role in promoting the differentiation of
naïve CD4+ T cells into mature Th1 effector cells and it also stimulates NK cells and
CD8+ T cells to produce IFN-γ (178). The IL-12 is also required for the development of protective innate and adaptive immunity in mice to intracellular pathogens such as 66 Listeria monocytogenes, and Toxoplasma gondi in mice (118, 375). The IL-12 positively regulates its own production via the induction of IFN-γ that primes monocytes and neutrophils for further IL-12 production. The IL-12 activates the Jak/STAT pathway and other signaling molecules such as the Src family protein tyrosine kinase LcK. Its production is inhibited by IL-10, IL-11, IL-13 and type I IFNs (387).
-IFNs. The IFNs were initially described as antiviral cytokines but they also act on cell
proliferation and differentiation and as activators of effector cells that compose the
immune system (304). They are classified into two types based on sequence homology,
the agents that induce their production, their cellular origin and their use of distinct
receptor systems. Both type I and type II IFNs stimulate CD4+ Th1 cells and actively inhibit Th2 cell activation (33, 109).
-Type-I IFN. Type I IFNs include the various IFN-α subtypes, IFN-β, -ε, -κ, -δ, -τ, and -
ϖ, which all function through the receptor components designated IFN-α receptor 1
(IFNAR1 and IFNAR2). They are produced by various cells such as leukocytes, fibroblasts, and pDC in response to virus and other infectious agents (106, 304). The
IFN-α/β signaling upregulates IFN-γ production by DC and T cells favoring the induction and maintenance of Th1 cells. They can also stimulate the expression of IL-15 by DC causing strong and selective stimulation of memory-phenotype CD8+ T cells.
Although many cells can produce IFN-α/β in response to viral and bacterial pathogens, pDCs are the most potent producers of these cytokines, and this production is further enhanced by stimulation of Toll-like receptors (TLR) on the surface of plasmacytoid DC and B cells, such as TLR7 and TLR9 and hypomethylated CpGs in bacterial DNA. The
IFN-α/β have effects on the development and function of B cells, enhancing B-cell 67 receptor (BCR)-dependent mature B2 cell responses and increase of survival and
resistance to Fas-mediated apoptosis (368). They also influence B cell responses either
indirectly through DC activation or directly by binding to the IFN-αβR expressed on B
cells, enhancing antibody responses and promoting Ig isotype switching (221).
-Type II IFN. Type II IFN (IFN-γ) acts through receptor components IFN-γ receptor 1
and 2 and is produced mainly by T cells, NK cells and cytotoxic T cells, but also DC, in
response to cytokines such as interleukin 12 (IL-12) (387). The IFN-γ production by
CD4+ Th1 cells is primarily induced by IL-12, especially during infections with
intracellular pathogens (242). The CD8+ T cells can also synthesize IFN-γ during viral
infections in response to IL-12 and IL-18 (188).
-IL-4. The IL-4 is produced in response to antigen activation by CD4+ Th2 cells, some
CD8+, NK1+ and γδ T cells. Mast cells also express IL-4 upon cross-linkage of the high
affinity IgE receptor, and could be an early source of IL-4 to naïve T and B cells during
initial antigen encounter. The IL-4 has an inhibitory effect on NK cell responses. The NK cells exposed to IL-4 are less cytolytic and produce low amounts of cytokines (245).
Antigen priming of naïve CD4+ T cells in the presence of IL-4 will initiate and amplify
their differentiation into a Th2 phenotype. The IL-4 also enhances B cell proliferation in
the germinal centers of secondary lymphoid organs and, in conjunction with signals
delivered through CD40L on mast cells, induces isotype switching to IgG and IgE (389).
-IL-13. The IL-13 is produced mainly by CD4+ Th2 cells. This cytokine plays a dominant role in resistance to most gastrointestinal nematodes. It modulates resistance to intracellular organisms such as Leishmania. It is also an important mediator of allergic inflammation directly contributing to airway hyperactivity. In humans, similarly to IL-4, 68 it promotes B-cell proliferation and class switching to IgG4 and IgE and also of
CD40/CD40 ligand costimulation. It also inhibits the production of pro-inflammatory mediators, such as prostaglandins, by monocytes and macrophages. However, unlike IL-4 this cytokine seems to be important in the effector phase of allergic inflammation, rather than in the initial differentiation of CD4+ T cells into Th3 cells (168, 400).
-IL-10. The IL-10 is produced by CD4+ CD25+ T cells with regulatory activity, and also
by dendritic cells, macrophages and B cells and it is able to inhibit cytokine production
by Th1 cells, and the induction of activities initiated by other cytokines such as IFN-γ,
IL-2, TNF-α, and IL-4. It can also be produced by γδ T cells and it might play a role in
maintaining the immune homeostasis in the gut (303). This cytokine also activates B cells
DNA replication and induces B cells to secrete IgM, IgA, and IgG (323). More recently,
a study showed that persistent lymphocytic choriomeningitis virus infection in mice
induces a significant upregulation of IL-10 by APCs, leading to impared T cell responses
and consequently to viral persistence (57). In mice, another subset of regulatory T cells
(CD8+ CD122+) produces IL-10 and can directly control CD4+ and CD8+ cells without
APCs intervention. They also suppress IFN-γ production and proliferation of CD8+ T
cells (97).
69 1.10 Mucosal immunity
The understanding of mucosal immunity is crucial for the development of
vaccines for mucosal pathogens, and only recently, the mechanisms of cell-cell
interactions, expression of co-stimulatory molecules, and T and B-cell homing have been
elucidated; however, much more remains to be determined.
1.10.1 Mucosal immune system
Inhaled or ingested soluble proteins and microbes enter the body through the
mucosal surfaces of the respiratory and gastrointestinal tracts. Therefore the mucosal
epithelia are the primary sites for antigen entry, constituting an important first line of
defense against invading pathogens.
The mucosa-associated lymphoid tissue (MALT) is widely distributed along the
mucosal surfaces and constitutes the initial inductive site for mucosal immunity. The
MALT is composed of several structures that are strategically located at sites that allow efficient antigen sampling from the mucosal surfaces. These structures include the conjunctiva-associated lymphoid tissue, the lacrimal drainage-associated tissue/duct- associated lymphoid tissue, the nose or nasopharynx-associated lymphoid tissue (NALT), the lymphoid tissues of Waldeyer’s ring, the larynx-associated lymphoid tissue, the
gastric mucosa-associated lymphoid tissue and the gut-associated lymphoid tissue
(GALT). Differences in distribution, occurrence, morphology, ontogeny and evolution of
MALT may vary between species (222)
Within the MALT, the lymphoid tissue is in close contact with the mucosal
surface and it consists of lymphoid tissue with lymphoid follicles and T-cell independent
70 interfollicular areas. The lymphoid follicles have germinal centers and mainly consist of
B lymphocytes surrounded by follicular dendritic cells, and CD4+ T lymphocytes and
macrophages. The interfollicular areas consist of CD4+ and CD8+ T lymphocytes. The
MALT structures have indirect contact with the lumen through an epithelium that is
infiltrated by lymphocytes and may also contain cells that are specialized in the uptake of
antigens termed membranous (M) cells (222).
1.10.1.1 NALT
Until recently, the NALT has only been described in rodents as a very small
(tonsil-like) aggregation of organized lymphoid tissue located on both sides of the nasopharyngeal duct dorsal to the cartilaginous soft palate, and it is considered analogous
to the Waldeyer’s ring in humans (349). In mice, the NALT appears only after birth,
reaching its maximal in size by 8 weeks of age (213). The pathway for the formation of
NALT tissue is still unknown but it seems to be independent of signaling involving the interleukine-7 receptor (IL-7R), lymphotoxin-β receptor (LTβR)-mediated signals (213).
Humans also have palatine, lingual and pharyngeal tonsils. However recently, a post-mortem study identified the presence of a morphologically distinct structure in
addition to the lymphoid structure of the Waldeyer’s ring as NALT in children (85).
-NALT in mice. In mice, follicular IgD+ cells are common in the follicular B cell areas,
together with some IgG2b+, IgA, or IgM+ cells, with a few IgM+IgA+ also being found in
these areas. Plasma cells that secret IgA are also detectable. In the parafollicular T cell
compartment of mice, the CD4+ cells are present in concentrations that are four times
higher thatn those of CD8+ cells. Most of these cells seem to be T helper 0 (Th0) cells
71 that could potentially differentiate into either Th1 or Th2 cells depending on the antigen
stimuli (213). In addition, atypical regulatory T cells with the phenotype
B220lowCD3lowCD4-CD8- c-kit+ and TLR2+ have been recently described in the NALT
(312), and might be the most abundant T cell population. In mice immunized IN with
antigen-filled liposomes, that had on their surface Sendai virus-associated fusion proteins, these particles were found in association with M cells, epithelial cells and macrophages in
the NALT and nasal airways. Specific serum IgG and IgA responses were also detected
in the nasal airway, saliva, gut and vaginal secretions (213).
Antigen-presenting cells such as dendritic cells and macrophages are also present in the
NALT (79). Other studies have also shown that IgA class-switching by IgM+B220+ B cells occurs in the NALT and that it seems to be an important site for the generation of B memory cells that can synthesize high-affinity IgA (333).
-NALT in swine. In swine the lymphoid tissues of the Waldeyer’s ring are well developed. In the nasopharynx there is a patch on the median roof forming the pharyngeal tonsil. Porcine also have tonsils of the soft palate and lingual tonsils (222).
The tonsils have high numbers of B lymphocytes, moderate numbers of αβ T lymphocytes and low numbers of CD8+ and γδ T lymphocytes compared to lymph nodes, peripheral blood lymphocytes and spleen (404).
-NALT in bovine. In the bovine, a moderate amount of lymphoid tissue is present in
Waldeyer’s rign. The oropharynx is protected by the lingual tonsil, the palatine tonsil and the tonsil of the soft palate. In the pharyngeal tonsils patches of microvillus-bearing cells
(most likely M cells) are found in the ciliated epithelium at 5 to 6 monts of gestation. And after birth, upon antigen contact, they become fully developed. The low number of 72 germinal centers and intraepithelial lymphocytes in those animals may be contributing
factors to their increased susceptibility to infections. With age, the size of the pharyngeal tonsils decreases (after 7 years of age), particularly the size of the lymphoid follicles, however ther is a relative increase in the proportion of T lymphocytes (222).
1.10.1.2 GALT
The Gut-associated lymphoid tissue (GALT) is separated from the intestinal lumen by a layer of epithelia that is formed predominantly by enterocytes interspersed by mucus-secreting goblet cells. The villus also contains scattered intraepithelial lymphocytes. The lamina propria is located below the villi. The portion underneath the follicle-associated epithelium that overlay the Peyers’ patches is called the subepithelial dome.
The GALT can be divided into inductive and effector sites. The Peyer’s patches, mesenteric lymph nodes and smaller isolated lymphoid follicles form the inductive sites.
The GALT effector sites contains lymphocytes distributed throughout the epithelium and the lamina propria of the mucosa (270).
GALT inductive sites:
-Peyer’s patches. The Peyer’s patches are lymphoid aggregates that are found along the
submucosa of the small intestine. Mature Peyer’s patches contain large B cell follicles interrupted by T cell zones. The tissue genesis of Peyer’s patches starts with the activation through the IL-7R or tumor-necrosis-factor-related activation-inducer cytokine of the CD3-CD4+CD45+ cells that are considered to be the common inducers of secondary lymphoid tissue. Upon activation, these cells express the lymphotoxin-α1β2
73 (LT-α1β2) heterotrimer, which then binds to the LT-β receptor (LT-βR) expressed on stromal cells. This induces signal transduction through the nuclear factor (NF-κB)- inducing kinase (NIK). The NIK then promotes the expression of adhesion molecules and/or chemokines. These homing molecules trigger the accumulation of lymphoid cells at the site of Peyer’s patches. Therefore, the IL-7R- and LT-βR-mediated signals are essential for the tissue formation of the Peyer’s patches (213).
The epithelium that separates the Peyer’s patches from the intestinal lumen has lower levels of digestive enzymes and a brush border that contains less glycocalyx and other glycoproteins (270). Below this layer is the follicle-associated epithelium that contains M cells.
The M cells are specialized cells that provide controlled transport of microorganisms and macromolecules from the intestinal lumen into the lymphoid follicles below. These cells have invaginated basolateral surfaces that form intraepithelial
“pockets” in which different antigen-presenting cells (APCs), including macrophages, dendritic cells and B cells can be located (243). These M cells do not express MHC class
II molecules, and therefore probably do not process antigens themselves but instead, they transport the antigen to the professional APCs located in the epithelium or in the dome area. Those APCs can then move to the T cell areas and/or B cell follicles where they can interact with naïve lymphocytes to generate primed effector lymphocytes that preferentially migrate to the gut. After the antigen is cleared, most effector cells die, and a subset of long-lived memory cells survive and can rapidly increase immunity after re- encounter with their specific antigen (270).
74 Different subsets of dendritic cells have been described in the Peyer’s patches of mice. They consist of “myeloid” DC characterized by CD8α-CD11b+ markers, the
“lymphoid” expressing CD8α+CD11b-, and large numbers of IL-10 producing DC characterized by CD8α-CD11b- that are located outside the organized lymphoid areas, mainly in the dome region (187).
In swine, the architecture of the Peyer’s patches is similar to that of humans.
There are around 11-26 patches that are distributed through the jejunum, with their number remaining the same throughout life (293). During the first days of life this lymphocyte population extends and organizes, but the final growth and organization of the Peyer’s patches is partially dependent on antigen stimulation (322). Within each patch there are B cell follicles that are separated by interfollicular areas where T cells predominate. The M cells have also been described in the epithelium overlaying the dome of the Peyer’s patches (119). Only one single large Peyer’s patch is present in the ileum and is populated mainly by B cells in the young animals. As they grow older the patch diminishes and transforms into many isolated patches containing similar numbers of B and T cells as the discrete patches. These discrete patches in the ileum probably function as inductive sites for T and B cell responses (322).
In bovine, the GALT if formed by patches in the jejunum, one patch in the ileum, one in the colon adjacent to the ileocecal opening, one in the proximal loop of the ascending colon, several small patches in the rectum along the anal ring and isolated lymphoid follicles in the small and large intestine. The GALT develops in the fetus and jejunal Peyer’s patches can be recognized in 5-month-old fetuses, with their number increasing during fetal life. It is estimated that between 24 to 49 jenunal Peyer’s patches 75 are found at birth and remain for the lifetime. In the Ileum, Peyer’s patches undergoe involution with age and are replaced by a few isolated lymphoid follicles in animals with over two years. The lymphocyte subpopulations of the jejunal and ileum Peyer’s patches do not differ during fetal development; however, at birth the differences become evident.
In the jejunal Peyer’s patches, many T lymphocytes (predominantly CD4+) are present within lymphoid follicles and the IgG- and IgA-mRNA expression is abundant, whereas in the ileum Payer’s patches, fewer CD4+ and CD8+ T lymphocytes occur in lymphoid follicles and interfollicular areas. Germinal centers develop only in jejunal peyer’s patches and colonic lymphoid tissue but not in the ileum Peyer’s patches (222).
-Mesenteric lymph nodes. The mesenteric lymph nodes (MLNs) of the small intestine are the largest lymph nodes in the body and are the first LNs to develop during embryogenesis. In mice their development requires the TNF superfamily cytokine, TNF- related activation-induced cytokine and its receptor.
The DCs from the MLNs of mice that were fed antigen produced IL-10 or TGF-β
and were shown to preferably stimulate antigen-specific CD4+ T cells to produce IL-10
and/or TGF-β. They were similar to the TR1 or TH3 “regulatory T cells” identified in the
GALT of mice that were fed tolerogenic doses of proteins and have been linked to oral
tolerance (140). The production of TGF-β by the regulatory T cells is probably involved
in immunoglobulin-class switching of B cells to IgA production. Another unique property
of the DCs in the MLNs is their ability to induce the expression of α4β7 integrin by naïve
T cells (352).
-Isolated lymphoid follicles (ILF). These are mononuclear cell aggregates that resemble
the Peyer’s patches or lymph nodes in the human intestine. They are located mainly in the 76 small intestine, but they can also be found in the colon. The IFLs are composed of B
lymphocytes that have a phenotype of B-2 lymphocytes (CD23+ IgMlow IgDhigh CD5-
CD11b-) and constitute 70% of the cellular population. The ILFs also have a smaller population of TCRαβ+ CD4+ and TCRαβ+ CD8+ T lymphocytes, CD11c+ DCs and IL-
7R+c-kit+cells (153, 233). The organization of the ILFs is similar to that of the Peyer’s patches, containing a loosely organized germinal center with peanut agglutinin (PNA)+ B lymphocytes and a follicle-associated epithelium containg M cells, but the IFLs lack the
T-lymphocyte zone. The IFLs contain M cells and have lymphocytes and antigen- presenting cells in close proximity, which could facilitate the interaction between antigens, APCs and lymphocytes. IgA-producing plasma cells have also been found among the ILF cellular population, indicating that the ILFs could stimulate IgA class switching (233).
GALT effector sites:
- Lamina propria. The lamina propria (LP) contains several types of DC, including a
particular subset of dendritic cells, named plasmocytoid dendritic cells, that was first
described in the lamina propria of mice and later in the colon of humans (34). These cells
have a relatively immature phenotype CD8α+B220+ and some studies have shown that
these cells may be involved in the induction of tolerance to intestinal antigens (250). It is
also probable that DCs that are loaded with antigen from the villus mucosa migrate from
the LP to interact with naïve lymphocytes mainly in the MLNs.
Most lymphocytes in the LP express a αβ-TCR together with one of the
conventional co-receptors, CD4 or CD8αβ. The lymphocytes in the LP of the small
intestine express more CD8αβ and the ones in the large intestine express more CD4, 77 similar to the T cells of lymph nodes and spleen (74). In contrast to the conventional
peripheral T cells, the T cell receptor (TCR) repertoire of the lymphocytes in the LP,
especially of the small-intestine is oligoclonal, indicating that the re-encounter with the
specific antigen in the gut would result in the expansion of selected clones (17).
The LP CD8+ cells also have cytotoxic T-lymphocyte activity and the CD4+ T
cells might be regulatory T cells responsible for maintaining the homeostasis and local
tolerance to environmental antigens. These cells produce cytokines such as IFN-γ, but also IL-4 and IL-10. (211). Some of the antigen-experienced LP lymphocytes might help local B cells to produce IgA, some experiments showed that total and specific IgA antibody responses are absent in mice that lack MLNs, with responses to parenterally administered antigens remaining intact (403).
In the porcine LP, T cells predominate in the villi with CD8+ T cells being
distributed under the epithelium and CD4+ T cells are located further down in the LP.
High numbers of eosinophils and mast cells are also present in the villi and crypts (380).
In newborn piglets, the LP CD2 T cells are negative for both CD4 and CD8 markers, but
the numbers of CD2+ CD4+ T cells increases greatly in the first week of life. The numbers
of CD2+ CD8+ T cells remain low increasing only by 7 weeks of age; however, the ratio
of CD4 to CD8 T cells always remain higher than one (379). After stimulation of porcine
T cells isolated from the LP with concanavalin A, high levels of cell death were detected
when compared with lymphocytes isolated from the spleen, and the transcription of IL-4,
but not of IL-2 was detected (23). Previous studies with human LP T cells showed that
these cells have the potential to produce high levels of both IL-2 and IL-4, depending on
the signal during activation (365). Taken together the data suggests that the primary
78 function of the gut environment in swine is to maintain the homeostasis by preventing the
expression of active T cell responses to antigens that are normally present in the gut.
The dendritic cells in the LP of swine express MHC class II, CD16, SwC3 and
CD45 on their surface and are potent stimulators of primary responses (164). The B cells
and plasma cells are found mainly in the crypts, and plasma cells are predominantly IgA+ and IgM+ but some IgG+ cells are also detected (58).
-Intraepithelial lymphocytes. The intraepithelial lymphocytes (IELs) express CD3+ and predominantly CD8αβ, and cells that express CD8αα exclusively (single positive) are also found. The TCRγδ+ IELs express CD8αα, whereas the TCRαβ+ IEL express either
form of CD8. In mice these TCRγδ+ cells are the most numerous. The IELs are located in
the basolateral membrane of the intestinal epithelial cells that form the epithelia overlying
the LP (74). Due to their location, these cells are one of the first to encounter antigens
that pass through the mucus layer on the luminal surface of the intestinal epithelia.
Although the function of these cells is still controversial, many in vitro assays have
shown that they have cytotoxic functions (NK activity, antiviral activity, anti-tumor
activity), helper T cell activity for antibody production and maintenance of oral tolerance,
amongst others (31).
In the pig, the IEL increases approximately 10-fold from birth to 2 months of age,
and in 2-month-old germ-free piglets the IEL numbers are around 6 times lower than the
the number in conventional pigs. In 45-day-old GF piglets the pattern of IELs cell
markers (20% CD8+, 7%, CD4+ and 60% CD2+) is similar to that of 5-day-old
conventional pigs.
79 In 9-month-old conventional pigs very few CD2+ T cells do not express CD4 or CD8
markers, reflecting the importance of the gut microflora in the increase of the IELs
numbers and their maturation (321).
1.10.1.3 IgA
The IgA is the most abundantly produced immunoglobulin (Ig) and is the main
antibody class at mucosal surfaces. In the serum it is the second most prevalent Ig. It
exists as two different subclasses (IgA1 and IgA2). The IgA1 has 13 extra amino acids in
the hinge region. The hinge region in IgA1 is a target for bacterial proteases, but IgA2 is
not susceptible to proteolysis by these proteases. The IgA1 predominates in the serum of
humans and it exists mainly in a monomeric form (10). In contrast, the IgA present in the
mucosa is produced as a dimer (dIgA) by local B cells and this dimerization is stimulated
through linkage with the J-chain peptide. The binding of dIgA to the polymeric Ig
receptor (pIgR) on the basolateral surface promotes the transport of the dIgA towards the
apical surface of the epithelial cells. After being transported, the pIgR is cleaved and the
dIgA is released but is still associated with the cleaved pIgR ectodomain called secretory component (SC). The complex of dIgA with SC is referred to as secretory IgA (sIgA)
(292). Its functions include inhibition of bacterial or viral adherence to the mucosal wall, prevention of invasion by microorganisms from the gut and neutralization of bacterial toxins and viruses. The IgA can also bind to antigens in the subepithelial and intracellular compartments and through its translocation across the epithelium it can actively excrete the antigen into the lumen (259). The FcαRI is the transmembrane receptor for IgA. It is expressed on cells of the myeloid lineage including neutrophils, monocytes,
80 macrophages, eosinophils and a subpopulation of DC. The expression of the FcαRI on the surface of neutrophils is upregulated by TNF-α and IL-8 and on the surface of monocytes by lipopolysaccharide, TNF-α and GM-CSF, whereas TGF-β, IFN-γ, and IgA
itself downregulate the FcαRI expression. The FcαRI is composed of two extracellular
Ig-like domains, a transmembrane region and a small cytoplasmic tail, and while the monomeric IgA has low to medium affinity for this receptor, the pIgA and IgA complexes have high avidity for it. Because the cytoplasmic tail of the FcαRI does not have signaling motifs, the receptor needs to associate with the FCRγ chain for signaling, and upon activation it mediates cellular functions such as endocytosis, phagocytosis, antigen presentation and release of inflammatory mediators (292).
1.10.1.4 T and B cell homing
The balance between local immune responses and mucosal homeostasis depend on the organized trafficking of memory/effector B and T cells. This migration of antigen- primed cells from inductive to effector sites seems to depend, at least partially, on the expression of adhesion molecules on the endothelial surface and their cognate integrins on lymphocyte surfaces (52).
In the NALT, the high endothelial venules (HEVs) express predominantly peripheral lymph node addressin (PNAD) and less MADCAM-1 (79). Afferent lymphatic vessels are absent and the efferent lymphatics drain into the cervical lymph nodes in the upper thorax (167). The B cell responses occur in the NALT and B cells can migrate to distant sites, as shown after the IN immunization of mice with polymer-grafted microparticles that elicited antigen-specific B cell responses. 81 These responses were mainly from IgA-secreting cells in the NALT and the
cervical lymph nodes. Antibody responses were also detected in the sera and were
predominantly IgG (167).
In contrast to the NALT, the HEVs in the GALT express MADCAM-1, and it is
believed that the migration of lymphocytes through the draining MLNs and back to the
LP depends on the interaction between molecules such as α4β7 on the surface of the cells
and their ligand MADCAM-1 on the endothelial surface (41). The accumulation of
lymphocytes in the MLNs requires the expression of both L-selectin and α4β7 integrin adhesion molecules. These adhesion molecules usually direct lymphocytes to enter peripheral and mucosal tissues, respectively, and their presence suggests that the MLNs constitute the midpoint between the peripheral and mucosal recirculation pathways (277).
Priming of lymphocytes by DCs in mucosal tissues increases the expression of two molecules that promote specific homing to the gut, α4β7-integrin and the chemokine
receptor CCR9. The CCR9 ligand is expressed on the epithelium and venular
endothelium of the small intestine and the ligation of CCR9 by CCL25 promotes
lymphocyte adhesion to the endothelium of the small intestine and helps to trigger α4β7
integrin-mediated adhesion of human T cells to MADCAM-1 (95).
It is thought that the lymphocytes that are primed in the Peyer’s patch exit through
the draining lymphatics to the MLNs, where they reside for further differentiation, before
they migrate into the blood stream through the thoracic duct and finally accumulate in the mucosa (277). However, studies in Peyer’s patches null mice indicate that the T cells might not necessarily migrate to the MLNs after initial activation in the Peyer’s patches.
82 It is more probable that the antigen presentation to naïve T cells occurs in the
MLNs themselves, by APC that bring the antigen there after being loaded with antigen in
the Peyer’s patches (238).
Recirculating naïve B cells enter secondary lymphoid organs from the blood to
sample for their cognate antigens. Within the lymphoid organs, the localization of the B
cells mainly in the B cell follicles depends on their expression of the chemokine receptor
CXCR5 and on the localized expression of its ligands CXCL13 by stromal cells in the follicles (271). Naïve B cells also express moderate amounts of CCR7, the receptor for
CCL21 and CCL19 that are expressed in T cell zones and, in the case of CCL21 in the follicles in lower concentrations.
1.11 Mucosal vaccines
Mucosal vaccines are promising because they can be administered directly to the site where the mucosal agent causes the infection, and also because of the trafficking of cells of the immune system between the local mucosal and systemic lymphoid tissues.
They are easier and safer to administer compared to injectable vaccines and have also
greater acceptability, especially by children (78). Oral vaccines constituted by live or live
attenuated pathogens administered by the oral route also provide effective development
of serum and generalized mucosal immune responses, they offer superior protection
against reinfection, inducing immunologic memory. However, to date few vaccines have
been licensed for mucosal administration due to the risk for the development of mutations
or revertants. Some of them are vaccines against polio, adenovirus, influenza virus and
cholera vaccines.
83 More recently, vaccines composed of non-replicating particles or nucleic acid constructs are being developed and tested and constitute promising alternatives to vaccines using live attenuated pathogens (285).
One of the disadvantages of the mucosal vaccination is the risk for induction of peripheral systemic tolerance or “oral tolerance”. This phenomenon can be explained by a diminished capability that an animal or individual would have to develop an immune response when re-exposed to the same antigen (170). To avoid this potential problem, adjuvants can be used in conjuction with these vaccines and will be reviewed in the subsequent section.
1.11.1 Adjuvants and antigen delivery systems for mucosal vaccines
1.11.1.1 Mucosal adjuvants
-CT and LT. The most commonly used mucosal adjuvants in animals are cholera toxin
(CT), the E. Coli heat labile enterotoxin (LT) and their mutants. Both CT and LT are composed of a homo-pentamer of cell-binding B subunits associated with a single toxic- active A subunit. This subunit enzymatically ADP-ribosylates the Gs protein of adenylate cyclase resulting in increased cAMP production in the affected cells (170). They are both powerful mucosal adjuvants potentiating the immunogenicity of most of the antigens with which they are associated. Their effects include increasing the permeability of the intestinal epithelium allowing a greater uptake of the co-administered antigen, enhancing antigen presentation by dendritic cells, macrophages and B cells, promotion of isotype switching in B cells resulting in increased formation of IgA and stimulatory and inhibitory effects on T-cell proliferation and production of cytokines.
84 Studies of murine and human DC and other antigen presenting cells showed that the use of CT up-regulates the expression of MHC/HLA-DR molecules, CD80/B7.1 and
CD86/B7.2 co-stimulatory molecules and also of CCR7 and CXCR4 chemokine receptors (98, 115). The CT can also induce DC to secrete IL-1β that in turn induces dendritic cell maturation (98). It is reported that CT induces primarily a Th2 type of immune response characterized by secretion of IL-4, IL-5, IL-6, and IL-10 mainly by
CD4+ T cells and also by production of IgA, IgG1 and IgE antibodies. In contrast, LT induces a balanced Th1/Th2 response. The main disadvantage of both mucosal adjuvants is that they are both too toxic for human use (170).
To overcome this issue, LT and CT mutants, derived from site-directed mutations
of LT and CT, have been developed and have shown reduced toxicity and improved
adjuvanticity when administered by the nasal route when compared to the oral route
(307). Mutants that have a single point mutation in the ADP-ribosylase active site (A1)
are significantly less toxic than the original toxins (91, 122). The mutant, LT (R192G)
has a single amino acid substitution, of arginine to lysine in the proteolytic cleavage site,
that conferred protein resistance to trypsin cleavage and reduced ADP ribosyltransferase
activity and toxicity in vivo, retaining its adjuvant activity (87). Other mutants of CT
and LT proteins have only the B subunits of CT (CTB) and of LT (LTB) and have been
used as an alternative to increase the target antigen immunogenicity. However, they
have proven to be less efficient than the holotoxins, but with their adjuvanticity being
increase by their coupling with the target antigen (120, 149).
85 In Gn pigs orally inoculated with a single dose of attenuated human rotavirus (Att
HRV) followed by two intranasal doses (250 μg per dose) of rotavirus-like particles (2/6-
VLPs) vaccines using a mutant E. coli heat-labile toxin (mLT) (5 μg) as adjuvant
conferred moderate to high protection rates against virus shedding and disease after
challenge, when compared to controls. This response was similar to pigs given 3 doses of
Att HRV (410). Pigs that received these vaccine regimes with 2/6 VLP seroconverted to
serum IgA antibodies and had the highest titers of intestinal IgA and IgG antibodies when compared to the pigs that received different vaccine regimens (22, 410).
Recently, the intranasal administration of rNV VLPs to mice induced high serum
antibody titers and fecal IgA antibody responses. The oral route of vaccination was also
effective after mLT (R192G) was administered as adjuvant (236). In another study, the
immune responses of Gn calves immunized with two or three doses of BoNoV VLPs in
conjunction with different adjuvants such as immunostimulating complex (ISCOM),
mLT (R192G) and oil adjuvant (ISA50V), and protection after challenge with the
homologous strain of BoNoV were evaluated. In that study, fecal IgA antibodies were
detected only in the calves vaccinated intranasally with rVLP and mLT (R192G) and
after challenge, partial protection with delayed and shortened diarrhea were also observed
in these calves (154).
-Cytokines and chemokines. Several cytokines and chemokines have been tested in
mice as mucosal adjuvants and different combinations of cytokines have shown to be
effective as nasal adjuvants (355). The administration of IL-1 in conjunction with Th1-
inducing cytokines such as IL-12, IL-18 and GM-CSF can be as effective a mucosal
adjuvant as CT, eliciting both Th1 (IFN-γ) and Th2-like (mucosal IgA) responses even to
86 poorly immunogenic peptides (351). A high concentration of cytokines is required when the proteins are administered in the soluble form due to their short half-life. A solution to this problem is to administer the cytokines as a gene encoded by a DNA vaccine. The
DNA vaccination ensures that the protein is produced for a period of several weeks.
However, the mucosal application of DNA vaccines still needs to be improved for application in large animals or humans (78).
-Pattern recognition receptor binding adjuvants. Various molecular structures present in microorganisms can be recognized by the pattern recognition receptors (PRR) present on the surface of macrophages and dendritic cells amongst others (78). The potential adjuvanticity of these molecules has been recently studied. One of these PRR ligand, lipopolysaccharide (LPS) from Gram-negative bacteria binds to CD14 on the surface of mononuclear cells and is then transferred to TLR4. Stimulation of TLR4 leads to the activation of transcription factors such as NF-κB and the downstream signaling pathway, resulting in the production of pro-inflammatory cytokines (IL-1 and TNF-α) and in the increased expression of MHC class II on the cell surface.
The muramyl dipeptide (MDP) (N-acetyl-muramyl-L-alanyl-D-isoglutamine) is a peptide derived from the cell wall of mycobacteria and is one of the constituents of
Freund’s complete adjuvant. The MDP binds to TLR2 and it has been tested as an adjuvant for mucosal (oral, intravaginal, intraduodenal) immunization of mice and rabbits. In one study MDP induced serum IgA and IgG responses and also fecal IgA antibodies when administered orally (100 mg) with ovalbumin (5 mg) to rabbits (32).
87 Mannan, a mannose that binds to mannose receptors on phagocytes is present on the cell wall of yeast and when given IN to mice, coupled to recombinant protein antigens, it enhanced the IgA, IgG1 and IgG2 antibody responses in the serum and IgA responses in mucosal sites (353).
Oligodeoxynucleotides containing CpG motifs (CpG DNA motifs) are a type of pathogen-associated molecular pattern (PAMP) and constitute another alternative to mucosal adjuvants. These CpG motifs are recognized by TLR triggering a signaling pathway that results in NFκB and other transcription factor activation, leading to the secretion of pro-inflammatory cytokines and in the up-regulation of the expression of co- stimulatory molecules activating the innate but also the acquired immune system.
Cytosine-phosphate-guanosine (CpG) motifs, in which the cytosine is unmethylated, can be found in bacterial DNA, but can also be synthesized (CpG ODN) and have adjuvant properties when administered in conjunction with an antigen. These motifs are recognized and bind to TLR-9, expressed by plasmacytoid dendritic cells and B cells, inducing the secretion of pro-inflammatory and Th1-inducing cytokines and also enhancing the expression of MHC and co-stimulatory molecules on the surface of antigen presenting cells (215). When administered IN with purified protein antigens, they promoted a mucosal Th2 response with production of IgA and a systemic Th1 response in mice (256, 257).
The CpG DNA generally stimulates B cells, NK cells, DC and monocytes/macrophages and the optimal motif for activation of human cells is GTCGTT
(163). The CpGs can also enhance innate immunity in the gastrointestinal tract mucosa.
88 When CpG ODN were administered intragastricaly they elicited local production of the CC chemokines RANTES, MIP-1α, and MIP1-β and of the CXC chemokine IP-10 in the stomach and/or intestine, in the absence of any antigen (170).
1.11.1.2 Mucosal antigen delivery systems
-ISCOM. It is both a delivery system for antigens and an adjuvant. It is a 40 nm cagelike structure consisting of subunits built from the interaction of saponins (most commonly the Quillaja saponin) that are surfactactants with a hydrophilic moiety, with lipid particles
(cholesterol and phospholipids) at a molar ratio of approximately 1:1. The hydrophobic interaction between these components and the antigen facilitates the assembly of the
ISCOM complex, and several copies of the antigen may be integrated (372). After administration by either parenteral or mucosal routes, ISCOMs are efficiently taken up by
APC. Generally, ISCOM enhances IFN-γ and IL-2, down-regulates IL-10 and does not enhance IL-4 responses. In mice, ISCOMs carrying influenza virus antigens enhanced
APC activity improving targeting and uptake of the antigen resulting in the production of pro-inflammatory cytokines (IL-1, IL-6), Th1 (IL-12) and in the increase of MHC class II expression and co-stimulatory molecules such as B7.1 and B7.2 on the surface of lymphocytes (267). However, responses may vary greatly, depending on the antigen used.
Gnotobiotic pigs immunized with one oral dose of AttHRV followed by two oral doses of 250 μg 2/6 VLP + 1,250 mg of ISCOM (VLPs containing VP2 from the bovine
RF strain and VP6 from virulent Wa HRV incorporated into the ISCOMs) had 75% protection rates against virus shedding and 50% protection rates against diarrhea after
89 challenge with the virulent WaHRV. These protection rates were similar to those of the
pigs that received three-dose AttHRV vaccine (185). This 2/6 VLP + ISCOM vaccine
regimen also boosted antibody titers and ASC responses after priming with AttHRV
(185, 279). The oral priming with AttHRV followed by boosting with two IN doses of
2/6 VLPs + ISCOM vaccine regimen was also evaluated in the Gn pig model. Pigs that
received three oral doses of AttHRV or one oral dose of AttHRV + 2 IN doses of 2/6
VLP + ISCOM had the highest protection rates against diarrhea after challenge with virulent WaHRV. This regimen also induced the highest virus neutralization antibody titers and the highest IgA antibody titers to HRV in the IC of Gn pigs prechallenge (PID
28), proving to be a safe and efficient alternative to multiple-dose oral vaccines (124).
Immunizations of non-human primates with ISCOM-based vaccines have also
proved to be highly immunogenic inducing antibody responses to a variety of antigens
such as influenza, measles, HIV-1, SIV and rotavirus. Antigen-specific proliferative T-
cell responses have also been detected in monkeys immunized with an ISCOM-based
influenza vaccine (327).
-Liposomes. This alternative delivery system is formed by aqueous suspensions of
microspheres that concentrate subunit protein antigens for delivery to the lymphoid
tissue. Various types of lipid mixtures can be used, resembling to some extent, the lipid
composition of the cell membrane. In the past, liposomes were mainly used for systemic
immunizations; however, more recently their administration by mucosal routes (oral and
IN) has been tested in animal models with the IN route inducing longer lasting immune
responses (372). Liposomes mainly target macrophages and the incorporation of
phosphatidylserine into the formulation renders a negative charge to their surface
90 increasing their uptake into the Peyer’s patches after oral administration (16), and since liposomes can bind the ganglioside GM1, this system constitutes a promising mucosal delivery system (159).
1.12 Immunity to caliciviruses
Early adult NV volunteer studies were based on clinical observations and revealed a high level of susceptibility to NV illness, and the existence of short-term homologous immunity. In one of these early volunteer studies, the antigenic relationship between NV and Montgomery County agents was established. Volunteers that had been initially infected by NV, were challenged with the Montgomery County agent and showed resistance to illness. In the same study NV and Hawaii were characterized as antigenically distinct agents since infection by either one did not protect against disease caused by the other agent administered 7 to 15 weeks after the first infection (399).
Long-term immunity was also evaluated in homologous rechallenge studies using the 8FIIa NV strain as inoculum. When 12 volunteers received the initial dose of the inoculum, 6 became ill and when all volunteers were re-challenged with the same inoculum 27-42 months later, only the same 6 volunteers became ill again. A third dose of inoculum was then given to 4 of the 6 volunters that became ill after the second challenge and only 1 became ill again. These findings confirmed that short-term immunity was induced by NV infection, and that a single dose of inoculum was not sufficient to confer long-term immunity to NV (297).
91 With the development of more sensitive antibody-detection assays (139, 239), the
antibody levels to NoVs in the general population could be finally determined. In one
study, the serological responses of teenagers was evaluated after they had been exposed
to a NV contaminated water supply, and no association was found between preexisting
antibody levels in the serum and susceptibility or resistance to illness (30), showing that
immunity to NoVs is complex and should not be determined only by serum antibody
levels and also suggesting that there might be host factors that facilitate infection or that
confer resistance to theses agents. Only many years later, were some of these factors
identified as the histo-blood group antigens and the secretor status of the individual (224,
318), as previously discussed in the “viral susceptibility and resistance” section of this
review.
It was soon established that detection of IgM was a marker of recent NV infection
and that an increase of 4-fold or more in the IgG or IgA-specific antibody titers determined seroconversion to that agent (126, 253). Johnson et al. showed that after
repetitive exposures, the antibody levels to NV become associated with protection, and
defined that the short-term homologous immunity lasts for approximately 6 months after
challenge, with some individuals still retaining low antibody titers even after multiple
challenges (200). Since most adults can be exposed multiple times during the course of
their lives, interpretation of antibody responses can be confusing since pre-existing
antibodies may have been elicited in previous infections with an antigenically related
virus. In order to distinguish current from previous infections, the antibody avidity was
evaluated, and it has been shown that the NoV-specific IgG antibodies have low avidity
during a primary infection (184). The avidity of antibodies could not be distinguished
92 between homologous and heterologous responses; however, a homologous blocking
response, but not a heterologous response was detected after infections with genogroup
II.4 NoVs (315). The secretory response to NoVs has also been evalulated and it has been suggested that the presence of NV-specific fecal IgA could be used to identify symptomatic disease, since high titers of fecal IgA before challenge correlated with clinical illness, but not with protection against infection or disease (288).
In more recent studies using saliva as a source of sIgA, there was a correlation between the presence of memory sIgA in uninfected susceptible individuals and protection against NV infection (224). In a different study, more than 80% of the NV infected volunteers had a 4-fold or higher increase in NV-specific salivary IgA and/or
IgG in the convalescent-phase saliva when compared to the levels in the sample collected during the acute phase of infection (264), confirming that mucosal immunity might be involved in the control of NV infection .
Cellular and humoral immunity after SMV (GII.2 NoV) challenge has recently been assessed, and contrary to NV, SMV infection was not dependent on histo-blood group secretor status. About 60% of the volunteers seroconverted to SMV with IgG- specific antibodies, mostly of subclass IgG1. Peripheral blood mononuclear cells
(PBMCs) secreted increased levels of IL-5, IFN-γ and IL-2 upon NoV VLP stimulation; increased IFN-γ and IL-2 levels were also detected in the serum of volunteers after SMV challenge, resulting in a predominant, but not exclusive Th1 response (223).
The immune response to HuNoVs has also been evaluated in some primate species. In one study, newborn monkey from the Macaca nemestrina species were orally inoculated with either the original fecal samples, from human origin, that were positive 93 for Toronto virus (GII NoV) or with the first passage of the same inoculum in other
Macaca nemestrina monkeys (357). Animals from both groups shed virus in their feces by RT-PCR and probe hybridization; however, IgG antibodies were only detected when antigens derived from rNV baculovirus, but not when recombinant Toronto virus antigens were used. The authors suggested that the antibody production could have been suppressed by the presence of maternal antibodies because the macaques were less than 6 months old. In another study, common marmosets, cotton top tamarins and rhesus macaques were orally inoculated with NV and cynomolgus macaques were inoculated with Grimsby virus (317). Short-term viral shedding was detected in animals, except for the cynomolgus macaques; however, NV or Grimsby virus-specific antibodies were only detected in the serum of one rhesus macaque that shed virus for 19 days. Specific IgA antibodies could not be detected in the saliva from any of the animals.
The antibody responses to BoNoV (GIII.2) were evaluated in Gn calves inoculated with one oral dose of the virulent BoNoV (CV186-OH) and challenged with the same inoculum after serial passage in Gn calves 20 days after the first inoculation
(154). Serum IgG antibodies were induced after one dose of the virulent inoculum reaching a geometric mean titer of 2,560 at post-inoculation day (PID) 20. Serum IgA antibody responses were also detected at PID 10 and 20, and a 2-fold increase was observed in one of the calves after challenge. Fecal IgG antibody responses were also induced after BoNoV inoculation and after challenge the IgG antibody titers increased 6- fold compared to those from pre-challenge sera. Fecal IgA antibodies were also induced after the first inoculation reaching the highest titers at PID 10, and did not increase after challenge.
94 None of the calves inoculated with the BoNoV had diarrhea or shed virus after
challenge which together with the antibody response data, indicated protection against
BoNoV infection.
1.13 Treatment and prevention
1.13.1. Treatment
To this date, there is no specific treatment for calicivirus gastroenteritis. The
general treatment for viral gastroenteritis is symptomatic, and the most common
complication to avoid is dehydration secondary to the disease. Therefore, oral and/or
intravenous fluid and electrolyte replacement are necessary, and usually sufficient to
correct liquid deficit and prevent the water deficit caused by numerous episodes of
vomiting and diarrhea (394). The severity and duration of abdominal cramps during NV
disease in adults was reduced after bismuth subsalicylate therapy, however this therapy is not recommended for infants and young children (108, 202).
-Antivirals. Nitazoxanide, a thiazolide with antiviral activity that targets cellular pathways involved in the synthesis of viral proteins and also has anti-bacterial and anti- protozoa activity has been tested in the treatment of NoV gastroenteritis in patients of at least 12 years of age. In this trial, adult patients that ingested 500 mg nitazoxanide twice daily for 3 days had a mean of 1 day in reduction in time to resolution of symptoms compared to those that ingested a placebo tablet (320).
95 -siRNA. Small-interfering RNAs mediate RNA interference (RNAi), a biologic mechanism that protects genomes and that, upon activation, can degrade exogenous and transgene RNAs, as well as endogenous mRNAs, in a sequence specific manner. After its first description in plants, this mechanism has been described in fungi, nematodes, flies, and more recently in human cells. (9, 325).
The basic mechanism of RNA interference consists of triggering of gene silencing by basically three types of extraneous RNA molecules: transgenes, double-stranded RNA
(dsRNA), or RNA derived from the transcription of developmental genes. The events downstream from the formation or introduction of dsRNA are common to all RNA- silencing mechanisms (9).
During RNA interference, transgenes or even transposons integrated into the genome of the host cell as either direct or inverted repeats are transcribed into aberrant mRNAs that are recognized by cellular RdRP and can be converted into dsRNA. The
RNA virus genomes are converted into a dsRNA replication intermediate by viral RdRP, developmental genes are then transcribed into mRNAs that fold into double-stranded intermediates, as precursor small temporal RNAs. The resulting dsRNA is further processed into small 21 to 25 nt RNA fragments by the Dicer complex composed of an
RNAse III enzyme (Dicer) and probably adapter proteins and an RNA helicase. The siRNAs with phosphorylated 5’ termini are recognized and incorporated into the RNA- induced silencing complex, probably comprising an adapter protein and an RNA helicase that unwinds the double-stranded siRNAs to enable the antisense strand to pair with target mRNAs which are then also cut into 21 to 25 nts fragments by an RNA endonuclease.
96 An RNA exonuclease may be responsible for complete degradation of target mRNA fragments. The stRNA are possibly incorporated into a translation-blocking complex comprising stRNA adapter proteins and an RNA helicase, to enable pairing with target mRNA of developmental genes to inhibit translation. Furthermore, in a silenced cell, the RNA-silencing signal, represented by the double-stranded aberrant RNA created by the RdRp or the siRNAs produced by Dicer could return to the nucleus to induce
RNA-directed DNA methylation or chromatin changes in the homologous genes (9, 96,
218).
The use of iRNAs to inhibit the production of viral proteins involved in the initiation or progression of diseases has the potential to become a novel and promising therapeutic approach. Since iRNA drugs could be designed to be more selective, they would be potentially more effective and less toxic than conventional drugs. However, unintended effects on gene expression could arise in the use of siRNAs-based drugs. A potential problem could be the activation of a subset of interferon genes by siRNAs leading to nonspecific translation inhibition of various proteins within the cell or within an organism. Another problem could be the recognition of siRNA by other mRNAs that have partial homology leading to degradation of unwanted mRNAs.
Recently, antisense oligomers targeting one of the three ORFs of the CV genus
Vesivirus significantly inhibited the replication of the isolates SMSV-13 and PCV Pan-1 in porcine kidney and African green monkey kidney cells. Various phosphorodiamidate morpholino oligomer sequences antisense to an upstream region of ORF1 have been tested and a single oligomer sequence was very effective in decreasing vesivirus titers up to 80%. This resulted in decreased cell death in both cell lines tested, in a dose-dependent
97 and sequence-specific manner showing that phosphorodiamidate morpholino oligomer could be potentially used as antiviral agents against calivirus infection (354).
However, because single-stranded RNA (ssRNA) viruses are highly prone to mutations, in order to avoid the emergence of siRNA-resistant variants the use of multiple conserved
RNA target sequences should be pursued. By repeatedly testing these siRNA in vitro and in animal models and making improvements in their design, the potential shortcomings could be resolved and the use of iRNA-based therapies could soon become a reality in treating human diseases.
-Hydrogels. Glycosylated hydrogels constitute another alternative prophylactic measure against NoV infections. In one study, the acrylic-group-conjugated human
HBGA type B was synthesized and combined with diallyldimethylammonium chloride (DADMAC) and acrylamide (Aam) to form the glycosylated poly(DADMAC)-poly(Aam) hydrogels. After polymerization, the hydrogels formed a mesh that was then submersed in an aqueous solution containing NoV VLPs, resulting in the entrapment of the VLP particles inside the hydrogel mesh. This system could be used as a potential oral antiviral drug, with the hydrogels containg the NoV particles being excreted from the body through the gastrointestinal tract (411).
1.13.2. Preventive measures
Since vaccines against HuNoV are still under study, preventive measures to avoid calicivirus infection include hand washing, ingestion of safe water and food and disposal of contaminated material to avoid spread of infection.
98 To prevent foodborne outbreaks, the proper cooling of foods, consumption of
cooked oysters, frequent handwashing and excused absence of sick personnel are
encouraged. To avoid waterborne outbreaks, the contamination of water sources with
vomit or feces containing calicivirus should be prevented, since NoVs can survive the
chlorine levels used in the treatment of public water systems (54). To avoid
contamination on board and spread of infection on cruise ships, the CDC recommends
that all basic food and water sanitation measures should be enforced and also the
immediate disinfection of ships during cruises, isolation of ill crew members and, if
possible, passengers for 72 hours after clinical recovery. Disinfection should be done
with freshly prepared chlorine solutions at concentrations of >1,000 ppm, phenol-based
compounds, and accelerated hydrogen peroxide products. Passengers and crew members
should also wash their hands with soap thoroughly and frequently and especially before
eating or drinking, after going to the bathroom and after shaking hands (5).
1.14 Vaccines
1.14.1 Animal studies
HuNoV are viruses that have a low infectious dose and are very resistant in the environment, and constitute a potential public health problem. These agents can affect
both undernourished as well as well nourished people being detected in people of all ages
worldwide, causing a great disease and economical burden. A vaccine would be of great
impact, especially as a preventive measure for the elderly, people that work in a confined
area or in very close contact with others. A few strategies have been developed and tested
99 over the last 2 decades, and since HuNoV are fastidious viruses, animals represent
important models in the development of vaccines against caliciviruses.
FCV whole inactivated virus or attenuated vaccines have been used to prevent
disease in cats for many years (311). However due to the great genetic variability of this
agent, these vaccines usually do not confer protection against infection and their
extensive use may sometimes result in the selection of vaccine resistant strains (84, 220,
311).
Alternative approaches to live or attenuated virus vaccines are being explored.
Vaccination of cats with three doses of a plasmid DNA containing the mature capsid protein gene of FCV strain F9, resulted in milder clinical symptoms and higher levels of antibodies when compared to controls vaccinated with only the plasmid lacking the FCV gene insert (344). The effect of baculovirus-expressed feline IFN-γ as a vaccine adjuvant has also been evaluated and it significantly increased antibody levels in the serum of cats vaccinated with inactivated F9 FCV strain, when compared to other commonly used adjuvants (331).
The VLPs derived from RHDV (VP60) were used to immunize BALB/c mice.
The animals received four doses (days 0, 7, 14 and 42) of a suspension of VLP+CT
(50µg/ml) either transcutaneously or intraperitoneally and sacrificed one week after the last immunization (408). Dendritic cells isolated from the bone marrow of the mice and cultured with rGM-CSF and VP60 VLPs secreted low concentrations of IL-12 in the supernant, whereas the addition of CpG to the DC/VLP co-culture induced high concentrations of IL-12, suggesting that the use of CpG DNA motifs could enhance the immune responses to the VLP vaccine. The antibody responses were also evaluated in the
100 mice sera and at PID 42, small amounts of antigen-specific IgG1 were detected in the
group vaccinated transcutaneously; however, in the group vaccinated intraperitoneally
larger amounts of IgG1 were produced. Splenocytes isolated at euthanasia were
restimulated in vitro with VLPs, and 72 hrs post-stimulation a high level of IFN-γ was
detected in samples from mice from both vaccination groups. No IL-5 was detectable in
either group. Vaginal lavages were also evaluated for the presence of IgA and mice
vaccinated intraperitoneally produced IgA early, peaking at PID 14, whereas the
transcutaneous route elicited low levels of IgA early that peaked late after several boosts
of antigen.
The NV rVLPs were used to immunize CD1 and BALB/c mice and elicited serum
IgG and intestinal IgA antibody responses. When the mucosal adjuvant CT was co-
administered with the rVLPs, higher levels of IgG antibodies were detected in the mice
sera, and the effect was dependent on the rVLP dose, with 200 μg of rVLP eliciting the
highest response. Thus, rVLPs were immunogenic when administered orally, even in the
absence of a mucosal adjuvant (26). The NV rVLPs were also administered IN to
BALB/c mice and were more effective in inducing serum IgG and fecal IgA antibody
responses, even at low doses (10-25 μg), when compared to the previous study. When the mLT (R192G) was used as the adjuvant, the responses to rNV VLPs were of higher magnitude and lasted longer than responses elicited only by the rVLPs (141).
Similar results were observed when GII (Dijon 171/96) rNoV VLPs were used as immunogen in BALB/c mice. The concentrations of IL-2, IFN-γ, IL-4, and IL-5 cytokines were assessed in cell culture supernatants at different time points after in vitro stimulation of cells from spleen, MLN and cervical lymph nodes with the Dijon VLPs. A 101 mixed Th1/Th2 pattern of secretion was detected with an early (post-stimulation day 3) peak of IL-2 when compared to IFN-γ; IL-4 and IL5 peaked 2 days later, suggesting that specific T lymphocytes homed from the site of induction to the intestine after IN immunization with the rVLPs (280). Periwal et al., showed that a modified cholera holotoxin (CT-E29H) enhanced both systemic and mucosal immune responses to NV rVLPs when orally administered to BALB/c mice, with high titers of IgG1 antibody in the sera. The adjuvant also increased the number of NV rVLP-specific IgA secreting cells in MNC isolated from Peyer’s Patches when compared to rVLPs alone. The addition of CT-E29H also increased the number of antigen-specific CD4+ T cells in the Peyer’s
Patches and both CD4+ and CD8+ T cell populations in the spleen measured by lymphoproliferation assay. Increased numbers of IL-4 antibody-secreting cells were also detected by ELISPOT, indicating that a stronger Th2 response was elicited in mice by the oral administration of NV rVLPs in combination with this non-toxic mutant of CT (301).
The oral administration of five doses of raw material from yeast cell lysates containing 0.1 mg of VA387 (GII.4 NoV) VLPs induced antigen-specific serum IgG in all 6 vaccinated mice and fecal IgA in 5/6 mice (401). Mice vaccinated with a higher dose (1 mg) developed higher and earlier responses with detectable serum IgG and fecal
IgA antibodies after the third administration of the vaccine, and both the serum and fecal antibodies blocked VA387 VLP binding to A, B and H human histo-blood group antigens.
Bovine norovirus rVLPs were also immunogenic in gnotobiotic calves inoculated
IN, only when mLT was given as adjuvant, eliciting fecal IgA antibody and conferring partial protection after challenge with virulent BoNoV (154).
102 Transgenic plants constitute a promising alternative for production and delivery of
vaccine antigens, being both potentially economically feasible and easy to deliver.
Transgenic tobacco and potato plants expressing the capsid protein of NV were created,
resulting in the formation of of VLPs identical to those expressed in insect cells. Both plants-expressing VLPs were immunogenic when orally administered to mice eliciting
both serum NV-specific IgG and secretory IgA antibodies.
A plum pox potyvirus (PPV)-based vector has also been used for the expression
of the VP60 structural protein of RHDV. Rabbits that were subcutaneously vaccinated
with extracts from VP60-expressing plants developed a strong immune response against
RHDV that resulted in protection against a lethal challenge with RHDV (107). In a
second study, transgenic potato tubers expressing VP60 were orally administered to
rabbits and only 2 out of 5 animals that received the highest dose of VP60 had detectable antibody titers after the third immunization and only the animal with the highest antibody titer survived the lethal RHDV challenge (251).
A different strategy used to obtain NV VLPs was to clone the NV capsid gene and use the Venezuelan equine encephalitis (VEE) replicon expression system (VRP-NV1).
In this system, the NV capsid gene was cloned into the VEE replicon expression vector and expressed in mammalian cells. After rVEE infection of baby hamster kidney cells, the capsid proteins self-assembled into VLPs. Mice inoculated with this construct developed systemic and mucosal immune responses to NV VLPs. A different construct
(VRP-NV2), containing three aa codon mutations from the original NV capsid protein was also engineered, but the resulting proteins failed to form VLPs.
103 When this mutant construct was inoculated into mice, it elicited reduced systemic
and mucosal immune responses when compared to VRP-NV1 (162), demonstrating that
intact VLPs may have important conformational epitopes.
Immunity during MNV-1 infection has also been investigated in mice. The virus was lethal to mice deficient in recombination-activating gene 2 (RAG2) (RAG/STAT1-/-) or to mice lacking signal transducer and activator of transcription 1 (STAT1-/-) but not to
RAG-/- or wild-type mice, revealing that STAT1 has an essential role in resistance to
MNV-1 infection and that B and T cell-dependent adaptative immunity are not required
for protection from lethal MNV-1 disease, suggesting that only innate immunity may be enough to confer resistance to this pathogen (207).
1.14.2 Human volunteer studies
The first vaccine to be tested in adult humans was a NV rVLP vaccine that was orally administered (100 or 250 μg) to healthy volunteers in two doses (day 1 and 21).
This vaccine did not cause any side effects and all volunteers that received the highest dose of vaccine seroconverted with IgG antibodies, and all had elevated IgG1 antibody responses, fifty-percent seroconverted with IgA antibodies, and a single dose was sufficient to elicit maximum immune responses in 85% of the volunteers. However, fecal
IgA antibodies could not be detected in any of the volunteers but they were detected in
the stool of 10 out of 15 volunteers who received the 250 μg dose. Thus, this first trial showed that NV rVLPs were immunogenic and constitute a potential alternative for a
HuNoV vaccine (25).
104 In a different study, Tacket et al., showed that all volunteers had a significant rise
in IgA ASC in blood following vaccination and booster 21 days after the first dose, regardless of the VLP dose (250μg, 500μg or 2000 μg) (361). In the group that received two doses of 250 μg of VLP, 90% seroconverted with serum IgG anti-VLP, with no significant different in seroconversion rates among the three dose groups. Serum IgA was observed in 90, 60, and 100% of the volunteers that received 250, 500 or 2000 μg of
VLPs, respectively; however, with lower titers than those for IgG antibodies.
Saliva, feces, vaginal wash and seminal fluid were also collected from volunteers for antibody detection. Forty-percent of 30 vaccinated volunteers developed anti-VLP salivary IgA, approximately one-third developed fecal or genital fluid anti-VLP-specific
IgA. No differences in specific fecal and salivary antibody titers were detected among the volunteers who received different VLP vaccine doses. However, 4 of the 6 volunteers who developed specific IgA antibodies in the vaginal wash fluid received the lowest VLP dose (250 μg). Cellular immune responses against NV VLPs were also evaluated in the
PBMC after in vitro antigen re-stimulation of the cells with the NV VLPs. Significant increases of IFN-γ were detected 21 days after immunization in the cells from volunteers who received the 250 and 500 μg of VLPs; no increases were detected in IL-4 production by the cells from any of the groups. No cell-mediated immune responses were detected in the volunteers who received the highest dose of VLPs (361).
These data show a dominant Th1-like response in the VLP vaccinated volunteers, that is in agreement with the previous results of Ball et al. (25) in adult volunteers vaccinated with NV VLPs and with the results for the naïve mice vaccinated with NV
VLP that also detected a bias towards a Th1 response (26). It is important to state that all 105 of these studies based their conclusions of a bias in Th1 on a predominat IgG2 antibody response and on the evaluation of few cytokine concentrations in the serum or in the supernatants of PBMCs collected from the blood of volunteers. Hence, before a predominant Th1 response is attributed to NV infection a more comprehensive evaluation of the immune responses to these viruses needs to be done with the assessment of a more complete panel of cytokines both systemically and locally, since HuNoV are gastrointestinal viruses, and local immunity may play an important role in protecting against infection and disease caused by these agents.
A recent study used alphavirus vectors to express the capsid protein of different
HuNoV strains (NV, DS, DF, HV, SMV, LV) in mammalian cells. The recombinant proteins from NV (GI/1), HV (GII/1), SMV (GII/2) and LV (GII/4) self-assembled into
VLPs and were used as antigens to evaluate homotypic and heterotypic antibody responses in adults that had previously been challenged with NV, SMV or HV. The sera collected during and up to 5 weeks after outbreaks of Desert Shield-like virus (DS),
Chiba-like virus (DF), and LV were also included in the study. The highest IgG response against NV VLPs, was detected in the sera of volunteers that had been previously infected with NV with a median fold-increase in IgG titer of 72 in comparison to pre- challenge sera, and heterotypic responses to NV VLPs were lower for all the other infection groups. Eighty-one percent of GI infected volunteers and 38% of GII infected volunteers seroconverted to GI antigens, and 35% of GI infected volunteers and 59% of
GII infected volunteers seroconverted to GII antigens (228).
106 These results suggest that a vaccine containing several HuNoV VLPs, rather than
a monovalent vaccine, could provide a more broad protection from various NoVs, since it
is thought that adults may come in contact with more than one ECV strain during the
course of their lives.
The novel approach of using transgenic plants expressing HuNoV antigens has
also been tested in humans. The vaccine consisted of potatoes
(Solanum tuberosum) that had been transformed with a binary vector that contained expression cassettes for NV capsid protein (NVCP) and kanamycin resistance resulting in the potato line NV140-13 that contains 4 copies of pNV140 T-DNA stably integrated into nuclear chromosomal DNA. The potatoes, containing NVCP and also NV VLPs, were propagated and the tubers harvested, and used as vaccine after being peeled. Adult volunteers were fed 2 or 3 doses of 150g of peeled raw potatoes that contained 215-715
μg of NVCP. Ninety-five percent of the volunteers who received 2 or 3 doses of the transgenic potatoes had significant increases in the numbers of IgA antibody-secreting cells isolated from PBMCs (25-115/106 PBMCs), and 20% of the volunteers developed
NV-specific serum IgG antibody (the geometric mean titer increased from 67 before to
757 after immunization) and 30% had NV-specific fecal IgA antibody. Most of the
volunteers in this study developed some type of immune response to the transgenic
potatoes, and possibly the immunogenicity of this vaccine could be improved by
selecting tubers that contained an elevated proportion of NV VLPs instead of only NVCP
and also by administering higher doses of these VLPs (360).
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152 CHAPTER 2
CYTOKINE AND ANTIBODY RESPONSES IN GNOTOBIOTIC PIGS AFTER
INFECTION WITH HUMAN NOROVIRUS GENOGROUP II.4-HS66 STRAIN
2.1 SUMMARY
A GII.4 human norovirus (HuNoV) HS66-strain infects and causes mild diarrhea
in gnotobiotic (Gn) pigs. In this study we evaluated systemic and intestinal humoral and
cellular immune responses to HuNoV-HS66 in orally inoculated pigs. Antibodies and
type-I IFN (IFN-α), pro-inflammatory (IL-6), Th1 (IL-12, IFN-γ), Th2 (IL-4), and Th2/T- reg (IL-10) cytokine profiles in serum and intestinal contents (IC) of the HuNoV-HS66 inoculated pigs and controls were assessed by ELISA at selected post-inoculation days
(PID 0-28). Using ELISPOT, we evaluated IgM, IgA, and IgG antibody-secreting cells
(ASC) and cytokine-secreting cells (CSC) in intestine, spleen and blood. In the HuNoV-
inoculated pigs, antibody titers in serum and IC were generally low and 65%
seroconverted. Pigs with higher diarrhea scores were more likely to seroconvert and
developed higher intestinal IgA and IgG antibody titers. The numbers of IgA and IgG
ASC were higher systemically than in the gut. In serum, HuNoV induced persisting
higher Th1 (low transient IFN-γ, high IL-12), but also low Th2 (IL-4) and Th2/T-reg (IL-
10), low, transient pro-inflammatory (IL-6) cytokine and notably a delayed IFN-α
153 response. Only intestinal IFN-α (early, late) and IL-12 (late) were significantly elevated
post-infection. Human NoV-HS66 also elicited higher numbers of Th1 (IL-12 and IFN-γ)
CSC when compared to Th2 (IL-4) and pro-inflammatory (IL-6) cytokine, with the latter
responses low in blood and intestine, reflecting low intestinal inflammation in absence of
gut lesions. These data provide insights into the kinetics of cytokine secretion in serum and IC of HuNoV-inoculated Gn pigs and new information on intestinal humoral and cellular immune responses to HuNoV that are difficult to assess in human volunteers.
2.2 INTRODUCTION
Noroviruses (NoVs) are the leading cause of foodborne illnesses in the U.S (22).
The NoVs are classified into 5 genogroups (I-V) and at least 27 genotypes. However, only strains from GI, with Norwalk virus (NV) as the prototype strain, GII and GIV have been reported to infect humans (18). The GII NoVs also occur in swine with GII.18
NoVs being genetically and antigenically similar to human strains, raising concerns for swine as potential reservoirs for GII NoVs (39). Recent increased worldwide outbreaks of
NoVs highlight a need for prevention and control measures including possible vaccines
(15, 29). However the lack of an animal model for HuNoVs and their failure to grow in cell culture monolayer hampers research on immunity and vaccines for HuNoVs.
Immunity to human caliciviruses (including HuNoVs) is complex and not completely understood. Early studies of human volunteers showed that serotype-specific short-term immunity is conferred by NV infection (16, 30, 43) and that not all individuals are susceptible to NV infection and/or disease. We recently showed that a subset of Gn pigs was susceptible to infection or disease after oral inoculation with the GII.4 HuNoV-
154 HS66 (8). Currently, two genetic factors (ABH histo-blood group antigens and secretor status) are associated with susceptibility or resistance to NV infection and disease in humans (14, 20). We further demonstrated a similar association between the phenotype
A+/H+ of Gn pigs and the development of diarrhea and higher rates of fecal viral shedding after infection with GII.4 HuNoV-HS66 compared to Gn pigs with non A+/H+ phenotype
(7). However, other investigators recently showed that in contrast to NV, Snow Mountain
Virus (SMV) (GII.2 HuNoV) infection was not influenced by histo-blood group or secretor status, suggesting that multiple factors may influence host susceptibility to the myriad of HuNoV genotypes (19).
The IgM and IgG antibody responses in serum and saliva of volunteers have been studied to assess immunity to HuNoV and at least a 4-fold increase in antibody titer has been considered as seroconversion (6, 16, 25). Fecal secretory IgA (sIgA) antibodies to
HuNoV have also been suggested as a marker for symptomatic disease (28). Although susceptible individuals who had memory sIgA antibody responses (indicated by NV- specific IgA antibody titers in pre-challenge saliva samples of Se+ individuals) were not infected by NV, some individuals who were susceptible to NV and who did not have strong salivary sIgA responses were also not infected (20). These findings suggest that multiple factors may be involved in susceptibility or resistance to infection and in the development of immunity to HuNoVs.
In swine, studies have shown that both humoral and cellular immune responses are important in resolving viral infections and also reveal the existence of a Th1/Th2 type of immune regulation like in mice and in humans (48). It is known that porcine IL-12 is similar to human IL-12, and that innate cells (dendritic cells and NK cells) produce IFN-
155 α and both innate (NK cells) and T cells produce IFN-γ after viral exposure (34, 42).
Porcine IL-6, produced by activated macrophages and other MNCs, is a pro- inflammatory cytokine, like in humans and it mediates and modulates the immune response, but it can also exacerbate inflammation. The IL-4 promotes development of helper T cells (Th2 cells) and induces division of B cells and their differentiation into plasma cells (26, 27) aiding in T cell dependent antibody production. The IL-10 may also be a regulatory cytokine in pigs as in humans functioning in the control of inflammation
(11).
In human adult volunteers exposed to SMV (GII.2 NoV), a dominant Th1 response with significant increases in serum IFN-γ and IL-2 during the acute phase of infection (PID2) was observed; however, local intestinal immunity and innate cytokine responses were not assessed (19). Thus, the aim of this study was to evaluate both intestinal and systemic antibody responses and cytokine profiles in Gn pigs after infection with GII.4 HuNoVs. To our knowledge, this is the first study to provide data on the immune responses of Gn pigs after GII.4 HuNoV infection, and also on intestinal immune responses to HuNoV, which is difficult to assess in human volunteers.
2.3 MATERIALS AND METHODS
2.3.1 Virus inoculum
A single aliquoted pool of the original human fecal sample identified as
NoV/GII/4/HS66/2001/US (HS66 strain) (8) was used for oral inoculation of Gn pigs using a dose of approximately 5.4 x 106 genomic equivalents (GE)/ml. Each Gn pig received one oral dose of the HuNoV inoculum consisting of 1 ml of the original 156 HuNoV-HS66 strain diluted 1:10 in minimal essential medium (MEM) (Gibco,
Invitrogen, Carlsbad, CA), that was further processed by vortexing, centrifugation at
3000 x g for 20 min and filtration through 0.8 μm followed by 0.2 μm filters. The Mock inoculum was MEM.
2.3.2 Inoculation of the experimental pigs
Near-term pigs were derived by surgery and maintained in sterile isolator units as previously described (23). Five-to-7-day-old Gn pigs were allocated into HuNoV-HS66- inoculated or mock-inoculated control groups. After receiving 8 ml of 100mM sodium bicarbonate orally to neutralize stomach acids, pigs were inoculated as follows: one oral dose (5.4 x 106 GE) of HuNoV HS66 strain (diluted and processed as described above to
a final volume of 10 ml) (n= 35 pigs), or equal volumes of MEM as controls (n=25). Of
35 HuNoV HS66-inoculated pigs, 3 pigs [two euthanized at post-inoculation day (PID) 2 and one at PID 21] did not show diarrhea or shed virus, nor did the older pig (PID21) seroconvert to HuNoV-HS66. These 3 pigs were excluded from the detailed studies of the immune responses leaving a total of 32 HuNoV-HS66 inoculated and infected pigs. Two additional pigs were inoculated with a fecal filtrate of HS66 virus inactivated with 0.01M binary ethylenimine (BEI) (inact-HS66), as previously described (8). The HuNoV-HS66 inoculated and control pigs were euthanized at PID 2 (HuNoV, n = 8; Controls, n= 5),
PID 6 (HuNoV, n = 5; Controls, n= 5), PID 8 (HuNoV, n = 5; Controls, n=5), PID 21
(HuNoV, n = 8; Controls, n=5), and PID 28 (HuNoV, n =9; Controls, n= 5). One of the pigs inoculated with the inact-HS66 was euthanized at PID 21 and the other at PID 28.
Four additional pigs that did not receive any inoculum were euthanized at 5 days of age, 157 and the results were used as baseline for detection of cytokines in the serum, cytokine secreting cell (CSC) and antibody secreting cell (ASC) ELISPOT assays.
2.3.3 Assessment of diarrhea
Daily rectal swabs were collected and diarrhea scores were noted and recorded
(0=normal; 1=pasty; 2=semi-liquid; 3=watery) from PID 0-6 as described previously (8).
Samples with scores 2 and 3 were considered diarrheic. The diarrhea cumulative score of each pig represents the sum of daily rectal swab scores from PID 1-6, and the mean cumulative score of each group is the sum of each pig’s diarrhea cumulative score divided by the number of pigs in that group.
2.3.4 Detection of viral shedding by RT-PCR
Viral shedding was determined using rectal swab fluids and 1:20 dilutions of intestinal contents (IC) by RT-PCR, using the primer pair Mon 431/433 (32) targeting the
RdRp region of HuNoV GII, using the same conditions as previously described (8).
However unlike as previously described, samples that were inhibited in RT-PCR, as
revealed by the use of an internal control (8), were re-tested after being re-extracted using
the RNeasy Mini kit (Qiagen Inc, Valencia, CA). Negative controls (rectal swabs from
mock inoculated pigs and RNAse-free water) for RNA extraction and RT-PCR were
included in each assay. A microplate hybridization assay (38) was performed to confirm
the product specificity using a probe specific for HuNoV-HS66
(PmonHS665’CTTGCTAATTTTGCTGTAGAATGATGGGCCGTGGA-3’).
158 2.3.5 Detection of viral shedding by antigen ELISA
The antigen ELISA was performed as previously described by Cheetham et al.
(8). Samples were considered positive when the mean absorbance (450 nm) of the
positive coating wells minus the mean absorbance of the negative coating wells was
higher than the mean absorbance of the negative control wells plus 3 times the standard
deviation.
2.3.6 Viremia
Sera from blood collected from pigs on PID 1-2 were analyzed by RT-PCR and
microwell hybridization for detection of HuNoV-HS66 RNA or amplicon, respectively,
as previously described (8). Unlike as previously described (8), samples that were
inhibited in RT-PCR were re-extracted using the RNeasy Mini kit and re-tested by RT-
PCR.
2.3.7 Antibody detection
An immunocytochemistry assay was performed to detect HS66-specific
antibodies in the serum and IC of Gn pigs, as previously described (44). For this assay a
recombinant baculovirus expressing HS66 capsid was used to infect Spodoptera
frugiperda (Sf9) cells as the HuNoV antigen source and the recombinant baculovirus infected cells or mock cells were subsequently fixed using 10% formalin in PBS. The
antibody titer was defined as the reciprocal of the highest serum dilution at which brown-
stained cells representing NoV antibody complexed to HS66-capsid antigen could be
detected.
159 2.3.8 Isolation of MNC for antibody and cytokine secreting cells ELISPOT
assays
Segments of the small intestine (jejunum and ileum), spleen, and blood were
aseptically collected at euthanasia and processed for the isolation of mononuclear cell
(MNC) populations, as previously described (37, 45). Single MNC suspensions from
each tissue and blood were prepared at concentrations of 5 x106 and 5 x105/ml in complete medium prepared with Roswell Park Memorial Institute (RPMI)1640 (GIBCO) enriched with 8% fetal bovine serum, 20mM Hydroxyethyl-Piperazine Ethanesulfonic
Acid (HEPES), 2mM L-glutamine, 1mM sodium pyruvate, 0.1mM nonessential amino
acids, 100 μg of gentamicin/ml, 100 μg of ampicilin/ml, and 50 μg of 2-mercaptoethanol.
2.3.9 ELISPOT assay for HuNoV HS66-specific antibody-secreting cells
(ASC)
An ELISPOT for detection of isotype-specific (IgM, IgA and IgG) ASC was
conducted using previously published methods (9, 46). Briefly, Sf9-cell plates infected with the HS66 recombinant baculovirus and non-infected Sf9-cell plates (Mock plates) were prepared and fixed as described in the antibody detection section and washed with deionized water prior to use. Single MNC suspensions from each tissue were added to duplicate wells (5 x105 and 5 x104 /well). Plates were then incubated at 37°C for 12 h in
5% CO2 and then washed three times with PBS buffer and incubated at 37°C for 2 h with
100 μl/well of HRP-labeled antibodies: goat anti-pig IgM (μ) (KPL) (0.25 μg/ml); IgA
(Serotec) (0.3 μg/ml); or IgG (KPL) (0.25 μg/ml). Plates were then washed three times in
PBS buffer and developed with TMB (KPL Inc.) for 2 h at Rt. The number of virus- 160 specific ASC were determined by counting blue spots in the wells, using a light
microscope, and were reported as the number of virus-specific ASC per 5 x 105 MNC, after any background spots (< 5), evident on the Mock plates, were subtracted.
2.3.10 Cytokine-secreting cell (CSC) ELISPOT assay
A cytokine ELISPOT for detection of IL-12, IFN-γ, IL-6, IL-4 and IL-10 CSC was performed as previously described (2) with minor modifications. Before being added to Multiscreen-IP sterile 96-well plates (Millipore, Bedford, MA) at concentrations of 5
x105 and 5 x104 /well, the cells were stimulated with 50 μg/ml of CsCl-purified HS66
virus-like particles (VLPs) (8) or 10 μg/ml of phytohemagglutinin (positive control) or
RPMI (negative control). Plates were then incubated at 37°C in 5% CO2 for 48 h. The
numbers of CSC were counted using an ImmunoSpot series 3A analyzer (Cellular
Technology Ltd., Cleveland, OH) and expressed as CSC per 5 x105 MNC. The HuNoV-
HS66-specific CSC numbers were computed after the numbers of CSC (< 4) in the
controls (RPMI-stimulated cells) were subtracted from the HS66 VLP-stimulated cells.
2.3.11 Cytokine ELISA assay
Blood was collected from pigs at PID 0, 1, 2, 4, 6, 8, 10, 12, 21 and 28, and intestinal contents were collected at euthanasia (PID 0, 2, 6, 8, 21, 28). Serum samples were processed and stored at -20° C (2). The IC samples were diluted 1:2 in MEM with a protease inhibitor cocktail to prevent cytokine degradation (1). The serum and IC were immediately frozen at –20°C until further testing. An ELISA test was performed to detect
IL-12, IFN-γ, IL-6, IL-4 and IL-10 as previously described (2). In addition to those 161 cytokines, an ELISA to detect porcine IFN-α was also developed. Plates were coated with rabbit anti-porcine IFN-α (0.75 μg/ml) (PBL, Piscataway, NJ), samples were added and then a monoclonal antibody (MAb) to porcine IFN-α (1.5 μg/ml) (PBL) followed by the addition of goat anti-mouse-HRP (5μg/ml). Standard curves were generated using recombinant porcine IL-12 (R & D Systems), IL-6, IL-4 (Biosource, Camarillo, CA),
IFN-γ, IL-10 (Biosource), and IFN-α (PBL). A computer-generated four-parameter curve-fit was used to calculate the concentration of each cytokine. The detection sensitivity limits for the reactions were as follows: 7 pg/ml for IL-12, IL-6, IL-4, IL-10 and IFN-γ; 15 pg/ml for IFN-α.
2.3.12 Statistical analysis
The cytokine concentrations, ASC (PID 6, 8, 21, 28) and CSC (PID 2, 6, 8, 21,
28) numbers were compared among and within groups using Kruskal-Wallis rank sum test (nonparametric). Spearman correlation coefficients were used to measure the correlations between diarrhea severity (cumulative scores) and the following immune responses: ASC or CSC numbers among intestinal and systemic tissues (spleen and blood) (PID 6, 8, 21 and 28) and HuNoV-HS66-specific convalescent serum and intestinal IgA and IgG antibody titers at PID 21 and 28. Statistical significance was assessed at P<0.05.
162 2.4 RESULTS
2.4.1 HuNoV-HS66 induces diarrhea, rectal shedding and viremia in Gn pigs
A detailed description of clinical signs, virus shedding and pathogenesis of
HuNoV-HS66 in gnotobiotic pigs was recently published (8). In the present study, a
different subset of pigs from the previous study was evaluated. Thirty-two of 35 (91%) of
the HuNoV HS66-inoculated pigs shed virus as detected by RT-PCR and microwell
hybridization. Only the 32 HS66-infected pigs were evaluated further in the immunologic
studies. Viral shedding was detected in the rectal swab fluids of all 32 pigs, by RT-PCR,
microwell hybridization and Ag-ELISA, from PID 1-6 (data not shown) with a mean
duration of shedding of 4 days (Table 2.1). Two of 5 pigs (40%) euthanized at PID 2 had
viral RNA detectable in the IC (data not shown). When shedding was determined by
antigen ELISA, 22/32 (69%) of the HuNoV-HS66-infected pigs tested had at least one positive rectal swab fluid sample during the acute phase of infection (PID 1-4) and 1 of 5
(20%) of the IC samples from pigs that were euthanized on PID 2 was positive by ELISA
(data not shown). As summarized in Table 2.1, of the HuNoV HS66-inoculated pigs,
27/32 (84%) had diarrhea, and 18/32 (56%) had viremia as detected by RT-PCR positive results in the serum.
2.4.2 Inact-HS66 does not induce diarrhea, rectal shedding or viremia in Gn
pigs
No viral shedding, viremia or seroconversion was detected in either of the two
Gn pigs that were inoculated orally with inact-HS66. Furthermore, no HuNoV HS66-
163 specific ASC was observed. The cytokine concentrations in the serum or IC and CSC
responses were also low and not significantly higher than Mock controls in any of the
tissues at any PID in the inact-HS66 group.
2.4.3 HuNoV-HS66 elicits low antibody responses in Gn pigs
Antibody titers in serum and IC as determined by immunocytochemistry were
generally low in the HuNoV HS66-inoculated pigs and ranged from 20 to 160 (data not shown). The IgM antibodies were initially detected in serum at PID 4. The IgA antibodies were initially detected in serum at PID 6 and peaked at PID 28 (geometrical mean titer
(GMT)=16). The IgG antibodies in serum were first detected at PID 21 and also peaked at PID 28 (GMT=14). Sixty-five percent of the HuNoV HS66-inoculated pigs seroconverted by PID 21-28 with either IgA (titers from 10 to 160) or IgG antibodies
(titers from 20 to 80). In IC, IgM antibodies were initially detected and peaked on PID 6
(GMT= 10). The IgA antibody response was first detected in IC and peaked at PID 6
(GMT=22), and IgG antibodies were initially detected at PID 21(GMT=7) and peaked at
PID 28 (GMT=11). The IC of nine of fourteen (64%) of HuNoV HS66-inoculated pigs tested had IgA and/or IgG antibody titers that ranged from 10 to 160. No HuNoV-HS66- specific antibody responses were detected in the serum or IC of the control pigs.
164 2.4.4 Seroconversion, serum and intestinal convalescent antibody titers to
HuNoV-HS66 are associated with diarrhea severity during the acute phase of
infection
When the titers of IgA or IgG convalescent antibodies in the serum or IC (PID 21
and PID 28) were compared with severity of diarrhea (cumulative scores) during
HuNoV-HS66 infection (PID 1-6), moderate but significant correlations were found
between diarrhea severity and convalescent serum IgA and IgG antibody titers (r=0.5;
p=0.004 and r=0.4; p=0.03, respectively) and the convalescent intestinal IgA and IgG
antibody titers (r=0.6; p=0.03 and r=0.7; p=0.01, respectively).
2.4.5 HuNoV-HS66 induced low numbers of HS66-specific ASC systemically
and locally in Gn pigs
The results of the ELISPOT assay for detection of virus-specific ASC in HuNoV-
HS66-inoculated and control Gn pigs are depicted in Figure 2.1. Overall the number of
ASC elicited in the HuNoV HS66-inoculated pigs was low, but most ASC responses
were significantly higher than in control pigs (Fig 1). The IgM ASC were initially
detected at PID 6 and peaked at PID 8 in both tissues and in blood (PBMCs), with the
highest numbers detected in spleen (20 ASC per 5 x 105 MNC). Significantly higher
numbers of IgA ASC compared to controls were also first detected at PID 6 in intestine
(peak at PID 6-8), and blood (peak at PID 6), and only later in spleen (PID 28). The IgG
ASC responses peaked later and the ASC numbers were significantly higher compared to controls only at PID 28 in intestine, at PID 21 and 28 in spleen and at PID 21 in blood with similar numbers of IgG ASC in both tissues and in the PBMCs.
165 2.4.6 Local ASC responses were strongly correlated with systemic ASC
responses after HuNoV-HS66 infection
At PID 6, high correlations were observed between intestinal IgA ASC and IgA
ASC in blood (r=1, p<0.0001); at PID 8 between IgM ASC in the intestine and in spleen
(r=0.9, p<0.0001), or in blood (r=1, p<0.0001) and between IgA in intestine and in spleen
(r=1, p<0.0001); at PID21 between IgG ASC in intestine and in spleen or in blood (r=0.8, p=0.0008 and r=0.9, p<0.0001, respectively); and at PID 28 between IgA ASC in intestine and in spleen (r=0.6, p=0.02) and IgG ASC in intestine and in spleen (r=0.8, p=0.0005) or in blood (r=0.7, p=0.003).
2.4.7 HuNoV-HS66 induced a balanced Th1/Th2 and a delayed type-I IFN
response in serum of Gn pigs
The cytokine ELISA results are summarized in Figure 2.2 and Table 2.2. The innate cytokine Type-I IFN (IFN-α) was detected in the serum of all pigs at all times tested including constitutively at PID 0. A low peak of IFN-α, although not significantly higher than controls, was detected in the serum of the HuNoV-HS66-inoculated pigs at
PID1. The IFN-α concentrations in the serum of the HuNoV-HS66-inoculated pigs, which were quantitatively the highest for any cytokine, were significantly higher than controls only later at PID 10 and PID 12 (2.2 and 1.6-fold increases over controls, respectively). The pro-inflammatory cytokine IL-6 was detected acutely in the serum of
HuNoV-HS66-inoculated pigs and was significantly higher than controls pigs at PID 2
(2.5 fold) and 4 (6.8 fold) coincident with both the diarrhea (PID 2-6) and viremia (1-2) periods and remained only slightly elevated until PID 10. 166 The HuNoV-HS66-inoculated pigs developed significantly higher Th1 (IL-12)
responses than controls (1.7 to 2.5-fold higher) at all PID except at PID 0, 6 and 28. The
low IFN-γ peak ocurred acutely (PID 2) in serum of the HuNoV-HS66-inoculated pigs, coinciding with the transient viremia at this time (PID 1-2). The IFN-γ concentration at
PID 2 was significantly higher (2.5-fold) compared to control pigs, but was transient remaining only slightly elevated through PID 10. Both IL-12 and IFN-α were also constitutively present at PID 0 and throughout PID 28 in the controls.
The Th2 (IL-4) and Th2/T-reg (IL-10) cytokines could only be detected in the serum of the HuNoV-HS66-inoculated pigs, but not in the controls, at each PID. The IL-4 and IL-10 concentrations were low, but were significantly elevated above controls at similar PID (4, 6, 8 and PID 2 for IL-4 only) with 2.5-7-fold increases over controls.
2.4.8 Only type-I IFN (early and late) and IL-12 (late) cytokine
concentrations were significantly elevated in the IC of the HuNoV-HS66 pigs
when compared to controls
The cytokine concentrations were measured in the IC of each euthanized pig and are presented as mean concentrations of each cytokine in the IC of each group, for each
PID that pigs were euthanized (Figure 2.3). All cytokine concentrations tested peaked early (PID 2, 6) in the HuNoV-HS66-infected pigs compared to controls, then peaked again at PID 21 or 28 for all but for IL-10 (Figure 2.3). Because of variability among pigs and possible degradation of some cytokine in IC, significant differences in cytokine concentrations in the IC of HS66-infected pigs, when compared to controls, were only observed for IFN-α at PID 2, 8, 21 and IL-12 at PID 28. However, the peak 167 concentrations of IFN-γ and IL-6 were higher in IC than in the serum (1.9 and 1.7-fold,
respectively) of the HuNoV-HS66-infected pigs, whereas those for IFN-α and IL-12
were higher in serum than in IC (4.1 and 4.7, respectively). In the IC, the significant peak
of IFN-α at PID 2 coincided with the period of viral shedding, viremia and diarrhea, and
in both serum and IC, a second and significantly elevated peak was detected at PID 10
and 21, respectively.
2.4.9 HuNoV-HS66 elicited higher numbers of Th1 (IL-12 and IFN-γ) CSC
when compared to Th2 (IL-4) and pro-inflammatory (IL-6) CSC both
systemically and locally
Higher numbers of CSC were generally detected at all times post-inoculation and
in all tissues and blood in the HuNoV HS66-inoculated pigs when compared to controls
(Figures 2.4 and 2.5). The Th1 (IFN-γ and IL-12) and Th2/T-reg (IL-10) CSC responses
were elicited at a much higher magnitude when compared to the pro-inflammatory (IL-6),
and Th2 (IL-4) responses (Figures 2.4 and 2.5). For Th1 CSC responses, IL-12 showed
divergent responses for both tissues and blood (Figure 4a). IFN-γ responses in the
intestine paralleled those in spleen. Significantly higher numbers of IFN-γ CSC were
detected and peaked acutely (PID 2) in intestine and spleen but peaked thereafter (PID 8)
in blood and were also significantly elevated later in intestine and spleen (PID 21 and 28)
and in blood (PID 21) when compared to controls. Significantly elevated numbers of IL-
12 CSC peaked first in intestine (PID 21) then in the spleen and blood (PID 28), and were
significantly higher than controls. When compared to Th1 responses, Th2 (IL-4) CSC
numbers were elicited at lower levels, and were only significantly higher than controls later in infection in intestine and blood (PID 21), and spleen (PID 28) (Figure 2.5). The 168 Th2/T-reg (IL-10) CSC numbers were higher overall than IL-4 CSC numbers, with the peak numbers detected later in infection in intestine (PID 21) and blood (PID 28)
although none were significantly higher when compared to controls. Peak numbers were
detected earlier in spleen (PID 8) when compared to intestine (PID 21) and blood (PID
28).
The pro-inflammatory (IL-6) CSCs were elicited at lower magnitude when
compared to all other cytokines tested (Figures 2.4 and 2.5), but were significantly
increased both early (PID 2, 6) and later (PID 28) in the blood and intestine of HuNoV-
HS66-infected pigs when compared to controls, with the significantly elevated blood
CSC responses closely mimicking those in the intestine (Figure 2.4).
2.4.10 Systemic HuNoV-HS66 CSC responses strongly correlated with local
HuNoV-HS66 CSC responses
At each PID with significant CSC numbers, we tested for correlations between
intestine, spleen and blood that might reflect trafficking of intestinal CSC stimulated
locally by HuNoV-HS66 infection through the blood or systemically. We found
correlations of CSC numbers between: pro-inflammatory (IL-6) CSC in the intestine and
in the spleen at PID 2 (r=0.9, p=0.0002) and PID 6 (r=1, p<0.0001) and between IL-6
CSC numbers in intestine and blood at PID 28 (r=0.9, p<0.0001). For Th1 (IFN-γ),
correlations were found between CSC in intestine and in spleen and blood, PID 8 (r=0.7,
p=0.04; r=1, p<0.0001, respectively), PID 21 (r=0.9, p<0.0001 for both) and PID 28
(r=0.9, p<0.0001; r=1, p<0.0001, respectively).
169 For Th2 (IL-4), correlations were found between CSC numbers in intestine and
spleen and blood at PID 28 (r=0.8, p=0.0005; r=0.8, p=0.0002, respectively), and also
between CSC numbers in intestine and blood at PID 21 (r=0.8, p=0.003).
Generally, local and systemic HuNoV-HS66 CSC responses strongly correlated indicating that HS66 induced both local and systemic cellular immune responses.
2.5 DISCUSSION
Because studies of immunity to enteric caliciviruses in any species are limited, evaluating B cell as well as T cell responses both locally (intestine) and systemically
(spleen and blood) are crucial for a better understanding of viral pathogenesis and host
immune responses. In this study, we assessed diarrhea, viral shedding, viremia and
seroconversion. We also evaluated the antibody titers in serum and IC and the ASC
numbers in intestine, spleen and blood. The innate type I IFN (IFN-α), pro-inflammatory
(IL-6), Th1 (IFN-γ and IL-12), Th2 (IL-4), and Th2/T-reg (IL-10) cytokine profiles were
also determined by ELISA in the serum and IC, and similar (except IFN-α, pig CSC
reagents are not available) CSC responses in intestine, spleen and blood were also
determined by ELISPOT in the Gn pigs inoculated with the GII.4 HuNoV strain or with
mock (controls).
A recent study conducted in our lab described the detailed pathogenesis of the HuNoV-
HS66 strain in gnotobiotic pigs (8). In the present study, using a new subset of pigs, only
the HuNoV-HS66-infected pigs (32 of 35 HS66-inoculated) as determined by rectal
shedding using RT-PCR and hybridization assay were studied further. In the infected
pigs, mild diarrhea was observed in 27/32 (84%) and viremia was observed in 18/32 170 (56%). Eleven of 17 (65%) of the pigs euthanized at PID 21 or 28 seroconverted to
HuNoV HS66 with either IgA or IgG antibodies, or with both. Our present results are in agreement with our previous study except that we found that 32/35 (91%) of the pigs inoculated with HuNoV HS66 had detectable viral RNA in at least one of the rectal swab fluid samples by RT-PCR and/or microwell hybridization assay in contrast to 44% of the pigs in our earlier study. This difference could be explained by the fact that all pigs in the
present study, but not in our prior study, received sodium bicarbonate prior to inoculation
in order to neutralize the acidic pH during viral passage through the pig’s stomach, as
previously described in human volunteer studies (12, 16). Second, we re-extracted and re-
tested each sample (from PID 1-6) that was inhibited in RT-PCR reactions and this
decreased the inhibition level of the samples and improved our detection sensitivity.
Overall, the antibody titers in serum and IC, as well as the number of ASC in
intestine, spleen and blood of HuNoV HS66-infected pigs were low. When we compare
our results to virulent-Wa HRV-infected Gn pigs (46), we observed that HRV also
induced an early low IgM ASC response in the small intestine that decreased by PID 21
similar to the HuNoV pigs. However, HRV induced approximately 10 times more IgA
and IgG ASC in the intestine of the Gn pigs at PID 21 than HuNoV-HS66 at PID 28, but
interestingly similar numbers of IgA and IgG ASC were detected in spleen and blood in
both HRV and HuNoV infected pigs at PID 21 or 28. The greater magnitude of the
intestinal ASC response in the HRV-infected pigs likely reflects the longer and more
pronounced intestinal viral replication and diarrhea seen, as well as the more extensive
intestinal lesions noted (40). The similar systemic ASC responses observed may reflect
the transient viremia evident in both.
171 In this study, significant positive correlations were found between intestinal and systemic ASC responses, including between IgA ASC in intestine and spleen and between IgG ASC in intestine and spleen and blood. Our findings are in agreement with the findings after virulent HRV infection of Gn pigs in which correlations were also found between IgA ASC numbers in the intestine and IgA ASC in the blood or spleen
(46). For both studies the correlation between IgA ASC from local and systemic tissues may reflect the trafficking of these cells between the gut lymphoid tissue and the systemic lymphoid tissues or blood (10).
Sixty-five percent of the HuNoV HS66-infected pigs that were euthanized at PID
21 or PID 28 seroconverted with either IgA, IgG or both, and coproconversion with either
IgA or IgG antibodies was detected in 9/14 (64%) of the IC from the HuNoV-HS66- infected pigs tested. We also observed moderate correlations between duration of
diarrhea and IgA and IgG antibody titers in the serum and in IC of the HuNoV-HS66-
infected pigs. Similar results were observed in studies that used primates as experimental
animal models where some animals that shed Toronto-like virus in the feces
seroconverted, whereas others did not (35). In another study, Rhesus monkeys that had clinical signs of infection and shed NV virus in the feces did not seroconvert, except for one that shed virus for a longer time (33), coinciding with our finding of positive
correlations between severity of disease and IgA and IgG antibody titers in both serum
and intestinal contents of HuNoV inoculated pigs. Our results suggest that the
development of antibody titers and diarrhea severity reflect intensity of intestinal
stimulation leading to an increase in local and systemic antibody titers.
172 Data from human adult volunteer challenge studies suggested that immunity to
HuNoV is variable and that the correlates of protective immunity to HuNoV are not
understood (12, 16, 19, 28). Short-term immunity to homologous virus has been shown consistently; however, the seroconversion rates vary (50-90%) (21). Very few studies have been done of local and secretory immune responses to infection with HuNoV (4,
28). In this study, although the convalescent IgA and IgG antibody titers detected in the
serum and IC of HuNoV-HS66-infected pigs were low, we found moderate positive
correlations between them and diarrhea severity during the acute phase of infection (PID
1-6). In early studies, fecal secretory IgA (sIgA) antibodies to HuNoV were also used as a marker for symptomatic infection (28). A more recent study failed to show a significant increase in convalescent fecal IgA antibody titers after oral exposure of volunteers to NV
(28). Local jejunal antibody titers were also measured to evaluate their role in resistance to NV infection in adult volunteers (13). Similarly, the authors did not find any correlation between prior jejunal antibody titers to NV and resistance to subsequent infection. More recently, 67% of SMV infected volunteers had a 4-fold or higher increase in salivary sIgA antibody titers to SMV after homologous challenge. It is important to emphasize that our animal model, the gnotobiotic pig, is a naïve animal that had not been previously exposed to any NoV or other microbe prior to HuNoV-HS66 inoculation.
Therefore the magnitude of the immune responses in these animals is predictably lower than that of adult human volunteers participating in HuNoV challenge studies who have probably been repeatedly exposed to NoVs during their lifetime. The presence of pre- existing antibodies to NoV elicited by previous infections with antigenically related viruses makes interpretation of the primary antibody responses confusing in such studies.
173 Thus, the Gn pig model allows assessment of the primary immune responses to HuNoV in a NoV-naïve animal model.
The production of Type I interferons is usually upregulated in the early phase of viral infections (5). In our study, an early, low but not significant (compared to controls) peak of type-I IFN (IFN-α) at PID 1 and a later but significantly higher peak at PID10 were observed in serum of the HuNoV-HS66-infected pigs. Significant increases in the concentrations of IFN-α in the IC of the HuNoV-infected pigs were also observed at PID
2, at PID 8 and later at PID 21 when compared to controls. We speculate that the earlier increase (PID 2) of IFN-α secretion in the IC was the result of a local, early response of the host’s innate immune system (epithelial and dendritic cells) to viral infection (31). It is possible that persistent antigen presentation after acute virus infection or persistence of viral genomic RNA inducing very low levels of transcription and translation of viral proteins, as suggested for VSV and influenza virus (36, 47), may also occur for NoVs stimulating the secretion of innate cytokines even after virus shedding was no longer detectable. This would explain the later peaks of IFN-α in the IC and in the serum after clearance of the acute viral infection.
Low to moderate levels of IL-6 (6-28 pg/ml) in serum of HuNoV-HS66-infected pigs were similarly detected in NoV-challenged humans by Lindesmith et al. (19), although levels of IL-6 in post-challenge human sera did not vary greatly from pre- challenge levels. Low numbers of IL-6 CSC, although statistically higher than the numbers in the controls, were also detected in the intestine, spleen and blood of HS66- infected pigs. The low IL-6 responses to viral infection could reflect the low virus load during virus replication and the limited pathology observed evoking only low 174 inflammation in the gut. In contrast to HuNoV infection of Gn pigs (since IL-6 levels
increased 1.5-6.8 fold over controls), IL-6 levels in the serum of HRV-infected Gn pigs
were higher and significantly elevated at PID 1-5 (17-57-fold higher than Gn controls)
reflecting the high virus load during replication in the gut and the extensive intestinal pathology induced (2).
In our study both early Th1 (IL-12 and IFN-γ) and Th2/T-reg (IL-4 and IL-10) cytokine responses were elicited in the serum of HuNoV-HS66-infected pigs. Early low
peaks of IL-12 (PID1-4), IFN-γ (PID2) and IL-6 (PID4) were detected in the serum of
HuNoV-HS66-infected pigs and were significantly higher than in controls (1.7-2.5, 2.5
and 6.8-fold increases, respectively). The early transient peak of IFN-γ observed in the
infected Gn pigs coincided with the early peak of IFN-γ in the serum of human volunteers
inoculated with SMV (GII.2 HuNoV strain) and based on our pig study coincides with
the peak viremia (19).
As shown in previous work done in our lab, the intestinal cytokine concentrations
are probably underestimated due to their instability to acidic pH and sensitivity to
proteolysis by intestinal enzymes. However, the cytokine levels in the IC of the HuNoV-
HS66-infected pigs were consistently higher than the levels in the IC of the controls.
Significantly higher numbers of IFN-γ CSC in the intestine, spleen and blood
during the acute phase of infection (PID 2, 6) corroborate the potential role of IFN-γ CSC
in stimulating CD8+ T cells that function in viral clearance during the immune response
to viral pathogens including curtailment of viremia and fecal virus shedding (17).
Significantly higher numbers of IFN-γ CSC were also detected later (PID 21 or 28) in
both tissues and blood most likely representing the pool of antigen-specific effector 175 memory Th1 lymphocytes at the local site of antigen encounter (gut) and also
systemically.
The IL-12 (Th1 inducer) was detected in the serum of pigs from both infected and
control pigs at each time point, with significantly higher levels both early and again later in the serum of the HuNoV-HS66-infected pigs. The early increases of IL-12 in the serum (starting at PID1), likely produced by macrophages and dendritic cells, and of
IFN-γ (PID2), potentially secreted by NK cells, may represent the early innate responses to viral infection. Later, significantly elevated serum and IC levels of IL-12 and numbers of IL-12 CSC were also detected in the HuNoV HS66-infected pigs compared to controls, first locally in intestine (PID21) and later systemically in spleen and blood (PID28). The
late peaks in the numbers of CSC in intestine, spleen and blood, with substantially higher
numbers of IL-12 CSC in systemic tissues when compared to the intestine may reflect the
pool of circulating antigen presenting cells (APCs) that secrete IL-12 after antigen-
stimulation (41) or the presence of Th1 effector cells secreting IL-12.
The Th2 (IL-4) and Th2/T-reg (IL-10) cytokines were detected in the serum of
HuNoV HS66-infected pigs at moderate to low levels at most PID. Both IL-10 and IL-4
were significantly elevated early (PID 2 or 4, 6, 8) in the HuNoV-HS66-infected pigs.
Numbers of IL-4 and IL-10 CSC were also low, but significantly higher numbers of IL-4
CSC were detected locally in intestine and systemically in spleen (PID 21), and again
later in spleen (PID 28). In studies of immunity to SMV in adult volunteers, with pre-
existing antibodies to NoVs, no significant changes were detected between pre and post-
challenge concentrations of IL-10 in the serum of adult volunteers or the ability of their
PBMCs to secrete IL-4 or IL-10 after in vitro stimulation with SMV (19). Furthermore,
176 because IL-10 is also considered to be a regulatory cytokine in pigs and humans (11, 24,
27), functioning in the control of inflammation (11), the low levels of IL-10 in serum and the lack of significantly elevated numbers of IL-10 CSC in HuNoV-HS66-infected pigs could be due to the low level of pathology (8) and inflammation induced by the GII.4
HS66 strain in pigs. This finding is also reflected by low very transient levels of the pro- inflammatory cytokine IL-6 in serum and low numbers of IL-6 CSC in intestine, spleen and blood in the HuNoV HS66-infected pigs.
We also found positive associations between local (intestine) and systemic (spleen and blood) IL-6, IFN-γ and IL-4 CSC numbers, suggesting their transient passage through the blood and spleen and homing back to the intestinal lamina propria of the Gn pigs as also shown for humans (3).
In conclusion, HuNoV-HS66 induced low levels of antibodies and low numbers of ASC both systemically and in the gut mucosa, and 65% seroconversion in pigs. In serum, HuNoV-HS66 induced higher Th1 but also Th2 responses. Type-I IFN responses were significantly elevated in serum of HuNoV-HS66-infected pigs later, at PID 10 and
12 compared to acutely at PID 2 in the intestinal contents during the viral shedding period. The low pro-inflammatory (IL-6) CSC responses coincided with the lack of pronounced diarrhea and histologic lesions (8) induced by HuNoV-HS66 in Gn pigs. This study provides data on the pattern and time progression of antibody and cytokine responses after GII HuNoV infection and further supports the previously described replication of HuNoV in Gn pigs (8).
177 To our knowledge this is the first study that comprehensively delineates the systemic and intestinal antibody, cytokine and CSC responses after HuNoV infection in an experimental animal model.
2.6 ACKNOWLEDGMENTS
We thank Dr. Ana Gonzalez for her review and suggestions for our paper and Drs.
L. Yuan and C. Lee for reviewing the manuscript. We also thank Dr. J Hanson, Mr. R.
McCormick and Ms. J. McCormick for animal care and Dr. J. H. Hughes, who kindly provided the original NoV GII.4 human fecal sample.
Salaries and research support were provided by state and federal funds appropriated to the Ohio State University. This work was supported by a grant from the
National Institute of Allergy and Infectious Diseases, National Institutes of Health, grant
No. R01-AI49742 to the corresponding author (L. J. Saif). Menira Souza was a fellow of
Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Brasilia,
Brazil, from July-2002 to July 2005.
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183 Inoculum Virus Diarrheac Diarrhea Viremiae Seroconversionf Coproconversiong # pigs sheddingb (%) (%) (%) (%) mean mean days mean cumulative scored days (range) (range) (range)
HS66 (n=32) 4Ag (1-6) 27/32 (84%)A 4 (2-6) 7A (2-15) 18/32 (56%)A 11/17 (65%)A 9/14 (64%)A
Control (n=25) 0B 1/25 (4%)B 0.3 3B (0-6) 0/25 (0%)B 0/10 (0%)B 0/10 (0%)B
aData is shown only for the 32 HuNoV-HS66 inoculated pigs that shed virus detected by RT-PCR; the 3 inoculated pigs that did not develop diarrhea, viremia or seroconvert are not included bMean number of days and range of shedding determined by RT-PCR cDiarrhea present if fecal swab scores were ≥2 after inoculation. dRepresents the sum of daily rectal swab scores from PID 1-6 of each pig divided by the number of pigs in that group eViremia was determined by RT-PCR on PID 1-2 184 fgSeroconversion and coproconversion were determined by immunocytochemistry (HS66 recombinant baculovirus infected Sf9 cell-staining assay) at PID 21 and 28 gValues in the same column with different superscript letters (A or B) differ significantly (p<0.05)
Table 2.1. Diarrhea, fecal virus shedding and viremia detected by RT-PCR and seroconversion detected by immunocytochemistry in Gn pigs inoculated with either HuNoV-HS66 or mock-inoculated controlsa
PID Cytokines 1 2 4 6 8 10 12 21 28 Innate IFN-α 1.9 1.3 1.4 1.5 1.4 2.2* 1.6* 1.1 1.1 Pro-inflammatory IL-6 1.5 2.5* 6.8* 1.8 1.5 1.8 1 1 1.5 Th1 IL-12 1.7* 1.7* 2.5* 1.8 1.7* 2.3* 1.9* 2.1* 1.4 IFN-γ 1 2.5* 1.3 1.3 1.3 1.3 1 1 1
185 Th2 IL-4 2.3 2.8* 5* 4.3* 7* 3.8 4 3.2 2.5 Th2/Treg IL-10 1.3 2.6 2.5* 2* 4* 1.5 1.5 1 1
a Mean fold increase at each PID * Statistically significant increases are bolded (p<0.05)
Table 2.2. Cytokine responses (fold increase over controls)a in serum of HuNoV-HS66-infected Gn pigs
◙ * * * * of gnotobiotic pigs Control bars are ◙ PBMC Spleen Intestine IgG ASC 0682128 0 6 8 21 28 0682128 5 0 30 25 20 15 10 5 0 30 25 20 15 10 5 0 30 25 20 15 10 * * , controls. * significantly higher than controls (P<0.05). □ * , HS66; ■ Post-inoculation day (PID) * * 0682128 Intestine Spleen IgA ASC 0682128 0682128 PBMC 5 0 30 25 20 15 10 5 0 30 25 20 15 10 5 0 30 25 20 15 10 * * * * * * IgM ASC not visible due to lack of response by controls the specific antigen. * * * inoculated with HuNoV-HS66 or Mock controls. Symbols: Figure 2.1.Mean numbers of isotype-specific (IgM, IgA and IgG) antibody-secreting cell responses in intestine, spleen PBMC 0682128 0682128 0682128 Intestine PBMC Spleen
5 0 5 0
30 25 20 15 10 30 25 20 15 10
5 0 ASC/5x 105 MNC MNC 105 ASC/5x ASC/5x 105 MNC MNC 105 ASC/5x 30 25 20 15 10 ASC/5x 105 MNC MNC 105 ASC/5x
186 600 50 500 40 400 30 IFN-α Innate Pro-inflammatory 300 * IL-6 20 * * 200 10 pg/ml * pg/ml 100 0 0 01246810122128 2 468101 2 21 28 Th1 200 01 180 IFN-γ 160 IL-12 14 140 12 * 120 10 100 * * 8 80 * * * * * 6 pg/ml 60 pg/ml 40 4 20 2 187 50 0 0 45 01246810122128 5 01246810122128 40 IL-4 Th2/T-reg 45 35 * 400 IL-10 30 35 25 30 25 20 * * 20 15 * * pg/ml pg/ml 15 10 10 * * 5 5 0 0 0 1 2 4 6 8 10 12 21 28 0 1 2 4 6 8 10 12 21 28 ♦ shedding ♦ shedding ♦ viremia ♦ viremia ♦ diarrhea ♦ diarrhea Post-inoculation day (PID)
Figure 2.2. Cytokine concentration in serum of gnotobiotic pigs inoculated with HuNoV-HS66 or Mock controls. Symbols: HS66; controls.
* significanltly higher than controls (P<0.05). ♦ duration period. 150 150 Innate 125 IFN-α 125 IL-6 Pro-inflammatory 100 * * 100 75 75 50 * pg/ml 50 pg/ml 25 25 0 0 02682128 02682128 Th1 50 IFN-γ IL-12 40 50 30 40 30 * 20 20 pg/ml
pg/ml 10 10 0 0 0 2 6 8 21 28 02682128 188 Th2/T-reg 50 IL-4 IL-10 40 50 30 40 30 20 20 pg/ml pg/ml 10 10 0 0 0 2 6 8 21 28 02682128 Post-inoculation day (PID)
Figure 2.3. Cytokine concentration in intestinal contents of gnotobiotic pigs inoculated with HuNoV-HS66 or Mock controls. Symbols:
■, HS66; □, controls. * significantly higher than controls (P<0.05). Results are expressed as the mean value of the small intestinal contents of all pigs
in each group. Pro-inflammatory CSC
Intestine Spleen Blood
IL-6 IL-6 30 IL-6 30 30 25 * 25 20 20 20 15 15 10 * 10 * * 10 * 5 5 0 0 0 * * CSC/5x 105 MNC 105 MNC CSC/5x 0 2 6 8 218 2 0 2 6 8 21 28 02682128
Th1 CSC 189 IL-12 * 400 400 400 IL-12 IL-12 300 300 300 * 200 * 200 200 100 100 0 0 100
CSC/5x 105 MNC 105 MNC CSC/5x 0 2 6 8 21 28 0 2 6 8 21 28 0 0 2 6 8 21 28 400 IFN-γ IFN-γ 400 IFN-γ 300 400 300 * * 300 200 200 * * * 200 * 100 100 * * * * 100 ** 0 0 0 CSC/5x 105 MNC 105 MNC CSC/5x 02682128 02682128 0 2 682128 Post-inoculation day (PID)
Figure 2.4. Pro-inflammatory (IL-6) and Th1 (IFN-γ and IL-12) mean cytokine secreting cell numbers in intestine, spleen
and PBMC of gnotobiotic pigs inoculated with HuNoV-HS66 or Mock controls. Symbols: ■, HS66; □, controls. * significantly
higher than controls (P<0.05). * 82128 biotic 6 2 02682128 IL-10 0 0 IL-4 400 300 200 100 0 60 50 40 30 20 10 * Th2 CSC , controls. * significantly higher than controls (P<0.05). □ Th2/T-reg Spleen Blood
02682128 Post-inoculation day (PID) , HS66; IL-4 ■ 0 0 2 6 8 21 28 60 50 40 30 20 10 IL-10 0 400 300 200 100 * Intestine 02682128 0 2 6 8 21 28 IL-10 IL-4 0
0
400 300 200 100 60 50 40 30 20 10
CSC/5x 105 MNC MNC 105 CSC/5x CSC/5x 105 MNC MNC 105 CSC/5x pigs inoculated with HuNoV-HS66 or controls. Symbols: Figure 2.5. Th2 (IL-4) and Th2/T-regulatory (IL-10) cytokine secreting cell mean numbers in intestine, spleen PBMC of gnoto
190 CHAPTER 3
PATHOGENESIS AND IMMUNE RESPONSES IN GNOTOBIOTIC CALVES
AFTER INFECTION WITH HUMAN NOROVIRUS NOROVIRUS
GENOGROUP II.4-HS66 STRAIN
3.1 SUMMARY
We previously characterized the pathogenesis of 2 host-specific bovine enteric caliciviruses (BEC), the GIII.2 norovirus (NoV) CV186-OH and the phylogenetically unassigned NB strain in gnotobiotic (Gn) calves. In this study we evaluated the Gn calf as an alternative animal model to study the pathogenesis and host immune responses to the human norovirus (HuNoV) GII.4 HS66-strain. The HuNoV-HS66 strain caused diarrhea
(5/5 calves) and intestinal lesions (1/2 calves) in the proximal small intestine (duodenum and jejunum) of Gn calves, with similar but less severe lesions than those described for the Newbury agent-2 (NA-2) and NB BEC. Viral capsid antigen was also detected in enterocytes of the jejunum and in macrophage-like cells in the lamina propria of the proximal small intestine by immunohistochemisty. All five HuNoV-HS66-inoculated calves shed virus in feces. Antibodies and cytokine (TNF-α, IL-12, IFN-γ, IL-4 and IL-
10) profiles were determined in the serum, fecal samples and intestinal contents (IC) of the HuNoV-HS66-inoculated calves (n=5) and controls (n=4) by ELISA in the acute and
191 convalescent stages of infection. The HuNoV-HS66-specific antibody (IgM, IgA and
IgG) and cytokine secreting cells (CSC) were quantitated by ELISPOT in mononuclear
cells of intestine, mesenteric lymph nodes (MLN), spleen, and blood at post-inoculation
day (PID) 28. Sixty-seven percent of the HuNoV-HS66-inoculated calves seroconverted with IgA and/or IgG antibodies to HuNoV-HS66, although antibody titers in serum and
IC were generally low (GMT=20-320). The highest numbers of antibody-secreting cells
(ASC) detected were IgG in intestine of the HuNoV-HS66-inoculated calves. The
HuNoV-HS66 strain induced Th1 (IL-12 and IFN-γ), Th2 (IL-4) and Th2/T-reg (IL-10) cytokines in serum and fecal samples, with significantly higher peaks of IFN-γ at PID 2, when compared to controls. The IL-10 was significantly higher in serum of the HuNoV-
HS66-inoculated calves increasing early at PID 2 in both the serum and fecal samples, concomitantly with the pro-inflammatory cytokine TNF-α. The TNF-α and IL-10
cytokines were elevated acutely in the IC of the HS66-inoculated calves and at PID 28 higher numbers of IFN-γ and TNF-α CSC were detected in MLN or spleen. The Th2 (IL-
4) and Th2/T-reg (IL-10) CSCs were also elevated in the intestine as were the IL-10
CSCs in spleen. Our study provides new data confirming HuNoV-HS66 replication and
enteropathogenicity in Gn calves and reveals important and comprehensive aspects of the
host’s local (intestine, MLN) and systemic (spleen and blood) immune responses to
HuNoV-HS66.
3.2 INTRODUCTION
Caliciviruses infect a variety of animal hosts, causing a wide range of diseases from gastroenteritis to fatal hemorrhagic disease (48). Human noroviruses (HuNoV) are
192 the leading cause of epidemic food and water-borne non-bacterial gastroenteritis
worldwide (16). The BEC have also been detected in cattle from England, Germany (13,
18) and the US (44). Two strains from Europe, Jena and the NA-2, are genetically similar to GI HuNoV (13, 29, 38) and constitute a third NoV genogroup (GIII) (38). The pathogenesis and immune responses of Gn calves to the bovine GIII.2 NoV CV186-OH and to the unassigned NB strain have been previously characterized in our lab (20, 44).
Both strains infected the villous epithelial cells of the small intestine, especially in the duodenum and jejunum and less in ileum, causing their destruction and resulting in
severe diarrhea. The HuNoV are fastidious viruses and only recently were GI and GII
HuNoV strains cultured in vitro in a complex organoid model of human small intestinal
epithelium (45). Therefore, because of the lack of routine in vitro culture assays for these
viruses, animals such as Gn pigs (10) and calves are important as infectivity models to
understand the pathogenesis and host immune responses to HuNoV in comparison to the
host-specific NoVs.
Immune responses differ according to the infectious agent and the cytokine
secretion pattern induced. Evidence of a polarized T cell response to certain pathogens
has been found in humans (7) and in mice (5). The TNF-α is a pro-inflammatory cytokine
that is produced by cells of the innate immune system, including
monocytes/macrophages, NK cells, mast cells, and neutrophils and it is an important
inflammation mediator having a broad spectrum of action including pathogen control and
induction of apoptosis (41). The Th1 cytokines (IFN-γ, IL-2) support macrophage
activation, generation of cytotoxic T cells and the production of opsonizing antibodies,
whereas Th2/T-reg (IL-4, IL-5, IL-10, IL-13) cytokines support B-cell activation, the
193 production of non-opsonizing antibodies, the control of extracellular parasites and the elicitation of allergic reactions (35). The Th1 responses occur during intracellular bacteria and viral infections, whereas Th2 cytokines predominate during parasitic infections, although this dichotomy is complex and in some infections both types of responses occur
(14). The IL-12 is mainly produced by macrophages, dendritic cells (DC), and other antigen-presenting cells (APCs). It is a key cytokine in the innate immune system, but is also produced in a T-cell dependent manner during the development of adaptive immune responses and it also stimulates NK cells and CD8+ T cells to produce IFN-γ (22). Type II
IFN (IFN-γ) is produced mainly by T cells, natural killer (NK) cells and cytotoxic T cells,
but also by DC in response to cytokines such as IL-12. The IFN-γ production by CD4+
Th1 cells is primarily induced by IL-12, especially during infections with intracellular pathogens (31).
The Th2 cytokine IL-4 is produced in response to antigen activation by CD4+
Th2 cells, some CD8+, NK1+ and γδ T cells. Mast cells also express IL-4 representing an
early source of IL-4 to naïve T and B cells during initial antigen encounter. The IL-4 also
induces isotype switching to IgG and IgE (32). The IL-10 is produced by T cells with
regulatory activity, and also by DC, macrophages and B cells. It inhibits cytokine
production by Th1 cells, and the induction of activities initiated by other cytokines such
as IFN-γ, IL-2, TNF-α, and IL-4 (39), exerting strong anti-inflammatory effects (25).
In cattle the Th1/Th2 paradigm is less defined, and antigen-specific CD4+ T cell
cells co-express IL-4, IFN-γ and IL-10 in response to pathogens such as Babesia bovis
and Fasciola hepatica (47), but in vivo responses to certain pathogens can be biased
towards a Th1 or Th2 response (8). The bovine IL-12 molecule is structurally similar to 194 that of humans and mice and it increases IFN-γ secretion in vitro by cattle peripheral
blood mononuclear cells (PBMC) in response to viral antigens (54). The IL-4 plays a role
in protective immunity in cattle against helminth parasistes (2). During persistent viral
infection, IL-10 was produced mainly by bovine monocyte/macrophages and during
Trypanosoma congolense infection, the increase in IL-10 synthesis coincided with decreased IFN-γ synthesis (46). The TNF-α is also a pleiotropic cytokine in cattle and it may induce apoptosis in B cells infected with the bovine leukemia virus, therefore playing an important role in the pathogenesis of this viral infection (23).
We previously confirmed that the HuNoV-HS66 strain infects Gn pigs and described its pathogenesis in Gn pigs (9, 10). We also have recently delineated the immune responses to infection by the HuNoV-HS66 strain in Gn pigs (M. Souza, S. M.
Cheetham, M. S. P. Azevedo, V. Costantini, and L. J. Saif, submitted for publication). In this study we report the enteropathogenicity, viral shedding, antibody and cytokine immune responses, both locally and systemically, of Gn calves experimentally infected with the HuNoV-HS66 strain, and document the replication of the HuNoV, not only in
Gn pigs but also in Gn calves.
3.3 MATERIALS AND METHODS
3.3.1 Virus inoculum
A single aliquoted pool of the original human fecal sample identified as
NoV/GII/4/HS66/2001/US (HS66 strain) (10), containing approximately 5.4 x 106 genomic equivalents (GE)/ml, was used for oral inoculation of Gn calves. Each Gn calf received one oral dose of the HuNoV inoculum consisting of 3 ml of the original
195 HuNoV-HS66 strain diluted 1:10 in minimal essential medium (MEM) (Gibco,
Invitrogen, Carlsbad, CA), that was further processed by vortexing, centrifugation at
3000 x g for 20 min and filtration through 0.8 μm followed by 0.2 μm filters. The Mock inoculum was MEM.
3.3.2 Inoculation of the experimental calves
Near-term calves were derived aseptically by Caesarean section and maintained in individual sterile isolator units (21). Five-day-old Gn calves were inoculated as follows:
one oral dose (1.6 x 107 GE) of HuNoV HS66 strain (diluted and processed as described
earlier to a final volume of 30 ml) (n= 5 calves), or equal volumes of MEM as controls
(n=4). The HuNoV-HS66 inoculated and control calves were euthanized one day after diarrhea was first observed, at PID 3 (HuNoV, n = 2; Controls, n= 2), and PID 28
(HuNoV, n =3; Controls, n= 2).
3.3.3 Assessment of diarrhea
Daily fecal samples were collected by digital anal massage using sterile gloves directly into sterile specimen cups. Diarrhea scores of feces were noted and recorded (0= normal; 1= semisolid; 2= pasty; 3= semiliquid; 4= liquid) as previously described (21).
Fecal samples with scores of 2 to 4 were considered diarrheic. The cumulative scores of each calf were calculated based on the sum of daily fecal sample scores from post- inoculation day (PID) 1-6, and the mean cumulative score of each group is the sum of each calf’s diarrhea cumulative score divided by the number of calves in that group.
196 3.3.4 Histopathology
Tissues of major organs (kidney, liver, spleen and lung) and 5 cm-long intestinal sections of the duodenum (~ 5 cm from the pylorous), jejunum (~ 5 cm into the intermediate jejunal zone), ileum (~ 10 cm from the ileocecal junction), mid-cecum and mid-colon were collected from controls and HuNoV-HS66-infected calves and fixed in
10% neutral-buffered formalin for 2 days. The preserved tissues were dehydrated in a
graded ethanol series, embedded in paraffin and transversely sectioned for staining using
Mayer’s hematoxylin and evaluated by histopathological examination using light microscopy.
3.3.5 Viral antigen detection by immunohistochemistry
For detection of HuNoV-HS66-viral capsid antigens, the tissue sections were prepared and cut as described above and collected on positively charged microscope slides (Fisher Scientific, Pittsburg, PA). Slides were kept at 60ºC for 20 min,
deparaffinized in xylene twice for 5 min, and rehydrated through a graded ethanol series
(100% to 50%). Antigen retrieval was performed using 100 µg/ml of proteinase K
(Invitrogen Corp.), and sections were immersed in 0.3% H2O2 in methanol for
endogenous peroxidase removal. The slides were washed 3 times in phosphate buffered
saline (PBS) pH 7.4 and blocked with 1% normal goat serum for 30 min at room
temperature (Rt). The tissues were immersed in either a 1:50 dilution of guinea pig hyperimmune antiserum against HuNoV-HS66 virus-like particles (VLPs) (10) or a 1:250
dilution of monoclonal antibody (MAb) NS14 (10), kindly provided by Dr. Mary Estes
(Baylor, College of Medicine, TX), that mapped to the P1 domain of the capsid protein of
197 all GII NoV tested (24). Tissues were incubated overnight at 4ºC. After washing twice in
PBS, tissues were incubated with the secondary antibodies, either horseradish peroxidase
(HRP)-labeled rabbit anti-guinea pig IgG (1:50) (Dako, CA) (for the primary guinea pig hyperimmune antiserum) or an alkaline phosphatase (AP)-labeled goat anti-mouse IgG
(1:200) (Dako, CA) (for the primary NS14 MAb). The tissues were incubated for 1 h at
37ºC, followed by immersing the sections in the substrate solutions: 3’-diaminobenzine
(DAB) (BD Biosciences, CA) for 10 min at Rt for the HRP-labeled secondary antibody; or a solution of red substrate (1 tablet of fast red in 2 ml of 0.1 M Tris-HCl, pH 8.2
(Roche Applied Science) for 20 min at Rt for the AP-labeled secondary antibody.
Sections were counterstained with Mayer’s hematoxylin and examined using light microscopy.
3.3.6 Detection of viral shedding by RT-PCR
Viral shedding was determined using rectal swab fluids and 1:20 dilutions of intestinal contents (IC) by RT-PCR, using the primer pair Mon 431/433 (42) targeting the
RdRp region of HuNoV GII, using the same conditions as previously described (10).
Samples that were inhibited in RT-PCR, as revealed by the use of an internal control
(10), were re-tested after being re-extracted using the RNeasy Mini kit (Qiagen Inc,
Valencia, CA). Negative controls (rectal swabs from mock inoculated calves and RNAse- free water) for RNA extraction and RT-PCR were included in each assay. A microplate hybridization assay (50) was performed to confirm the product specificity using a probe specific for HuNoV-HS66 (10).
198 3.3.7 Detection of viral shedding by antigen ELISA
The antigen ELISA was performed as previously described (10). Samples were
considered positive when the mean absorbance (450 nm) of the positive coating wells
minus the mean absorbance of the negative coating wells was higher than the mean
absorbance of the negative control wells plus 3 times the standard deviation.
3.3.8 Viremia
Sera from blood collected from calves on PID 2 were analyzed by RT-PCR and
microwell hybridization for detection of HuNoV-HS66 RNA or amplicon, respectively,
as previously described (10). As described for the fecal samples, serum samples that were
inhibited in RT-PCR were re-extracted using the RNeasy Mini kit and re-tested by RT-
PCR.
3.3.9 Antibody detection
An immunocytochemistry assay was performed to detect HuNoV-HS66-specific
antibodies in the serum and IC of Gn calves, as previously described (51). For this assay
a recombinant baculovirus expressing HS66 capsid was used to infect Spodoptera
frugiperda (Sf9) cells as the HuNoV antigen source and the recombinant baculovirus infected cells or mock cells were subsequently fixed using 10% formalin in PBS.
Antibody titers were defined as the reciprocal of the highest serum dilution at which
brown-stained cells representing NoV antibody complexed to HS66-capsid antigen could
be detected.
199 3.3.10 Isolation of MNC for ELISPOT assays to detect antibody and cytokine
secreting cells
Segments of the small intestine (jejunum and ileum), mesenteric lymph nodes
(MLN), a section of the spleen and blood were collected at euthanasia and processed for
the isolation of mononuclear cell (MNC) populations, as previously described (49, 52).
Single MNC suspensions from each tissue and blood were prepared at concentrations of 5
x106 and 5 x 105 MNC/ml in complete medium prepared with Roswell Park Memorial
Institute (RPMI) 1640 (GIBCO) enriched with 8% fetal bovine serum, 20mM
Hydroxyethyl-Piperazine Ethanesulfonic Acid (HEPES), 2mM L-glutamine, 1mM sodium pyruvate, 0.1mM nonessential amino acids, 100 μg of gentamicin/ml, 100 μg of ampicilin/ml, and 50 μg of 2-mercaptoethanol (E-RPMI).
3.3.11 ELISPOT assay for HuNoV HS66-specific antibody-secreting cells
(ASC)
An ELISPOT for detection of isotype-specific (IgM, IgA and IgG) ASC was conducted using previously published methods (11, 53). Briefly, Sf9-cell plates infected with the HS66 recombinant baculovirus and non-infected Sf9-cell plates (Mock plates) were prepared and fixed as described in the antibody detection section and washed with deionized water prior to use. Single MNC suspensions from each tissue were added to duplicate wells (5 x105 and 5 x104 MNC /well). Plates were then incubated at 37°C for 12
h in 5% CO2 and then washed three times with PBS buffer and incubated at 37°C for 2 h
with 100 μl/well of HRP-labeled antibodies: goat anti-bovine IgM (μ) (Serotec) (0.25
μg/ml); sheep anti-bovine IgA (Serotec) (0.3 μg/ml); and goat anti-bovine IgG (KPL) 200 (0.25 μg/ml). Plates were then washed three times in PBS buffer and developed with
TMB (KPL Inc.) for 2 h at Rt. The number of virus-specific ASC were determined by counting blue spots in the wells, using a light microscope, and were reported as the number of virus-specific ASC per 5 x 105 MNC, after any background spots (< 3), evident on the Mock plates, were subtracted.
3.3.12 Cytokine-secreting cell (CSC) ELISPOT assay
A cytokine ELISPOT for detection of pro-inflammatory (TNF-α), Th1 (IL-12 and
IFN-γ) and Th2/T-reg (IL-4 and IL-10) CSC was developed. Multiscreen-IP sterile 96- well plates (Millipore, Bedford, MA) were coated with monoclonal antibodies (MAb) to bovine TNF-α (5 μg/ml) (Bovine TNF-α screening set, Endogen; Pierce Biotechnology,
Inc, Rockford, IL); bovine IL-12 (2μg/ml), bovine IFN-γ (1.3μg/ml) (Bovine IFN-γ screening set, Endogen; Pierce Biotechnology); bovine IL-4 (7.5μg/ml) (Bovine IL-4 screening set, Endogen; Pierce Biotechnology) or bovine IL-10 (2μg/ml) (Serotec) diluted in coating buffer (0.05 M carbonate buffer, pH 9.6) and incubated at Rt overnight.
The plates were blocked with E-RPMI before MNCs, stimulated with 50 μg/ml of CsCl- purified HS66 VLPs (10) or 10 μg/ml of phytohemagglutinin (positive control) or RPMI
(negative control), were added to the plates at concentrations of 5 x105 and 5 x104
MNC/well. Plates were then incubated at 37°C in 5% CO2 for 48 h. After the plates were washed with PBS-0.05% Tween (PBS-Tween 0.05%), biotin-labeled detection Mabs to bovine TNF-α (2 μg/ml) (Bovine TNF-α screening set, Endogen; Pierce Biotechnology); bovine IL-12 (2 µg/ml) (Serotec); bovine IFN-γ (0.3 μg/ml) (Bovine IFN-γ screening set,
Endogen; Pierce Biotechnology); bovine IL-4 (5 μg/ml) (Bovine IL-4 screening set, 201 Endogen; Pierce Biotechnology) or bovine IL-10 (2 µg/ml) (Serotec) were added and the plates were incubated at 4º C overnight. Plates were washed and HRP-conjugated streptavidin (Biosource, Camarillo, Ca) was added at a concentration of 0.3 µg/ml, and the plates were incubated at Rt for 2 hrs. The spots were developed with AEC substrate
(Sigma, Aldrich, St. Louis, MO) and the numbers of CSC were counted using an
ImmunoSpot series 3A analyzer (Cellular Technology Ltd., Cleveland, OH) and expressed as CSC per 5 x105 MNC. The HuNoV-HS66-specific CSC numbers were computed after the numbers of CSC in the controls (RPMI-stimulated cells) were subtracted from the HS66 VLP-stimulated cells.
3.3.13 Cytokine ELISA assay
Fecal samples collected at PID 0, 1, 2, 4, 6, 10, 14 and 21; blood samples collected from calves at PID 0, 2, 4, 7, 10, 14, 21 and 28; and intestinal contents (IC) collected at euthanasia (PID 3 and 28) were tested. Serum samples were processed and stored at -20° C (4). The fecal samples and IC samples were diluted 1:2 in MEM with a protease inhibitor cocktail to prevent cytokine degradation (3), and were immediately frozen at –20°C until further testing. An ELISA test was performed to detect TNF-α, IL-
12, IFN-γ, IL-4 and IL-10. Briefly, 96-well microtiter plates (Nalgene, Nunc, Rochester,
NY) were coated using the same antibodies used in the ELISPOT assay but with different concentrations only for the MAb to bovine IL-12 (4 µg/ml) and the MAb to bovine IL-10
(4 µg/ml) (Serotec). Plates were incubated at Rt overnight and blocked with PBS-4% bovine serum albumin (BSA)-5% sucrose for 2 hrs at Rt before the samples were added.
After 2hrs at Rt, the plates were washed and the same detection antibodies were added at 202 the same concentrations used in the ELISPOT, except for the Mab to bovine IL-12 (4
µg/ml) and the Mab to bovine IL-10 (4 µl/ml) (Serotec). After 1.5 hr at Rt, the plates
were washed and the HRP-conjugated streptavidin (Biosource, Camarillo, Ca) was added at a concentration of 0.1 µg/ml. The plates were incubated at Rt for 1 hr and developed
with TMB (KPL Inc.). Standard curves were generated using recombinant bovine TNF-α,
IL-4, and IFN-γ (Bovine screening set, Endogen, Pierce Biotechnology) and recombinant human IL-12 and IL-10 (Biosource). A computer-generated four-parameter curve-fit was used to calculate the concentration of each cytokine. The detection sensitivity limits for the reactions were as follows: 7 pg/ml for IFN-γ, TNF-α and IL-4; 15 pg/ml for IL-10 and
IL-12.
3.3.14 Statistical analysis
The cytokine concentrations in the serum (PID 0, 2, 4, 7, 10, 14, 21 and 28) and fecal samples (0, 1, 2, 4, 6, 10, 14 and 21) and the isotype-specific (IgM, IgA and IgG) antibody titers were compared among and within groups using Kruskal-Wallis rank sum test (nonparametric). The percentage of calves with diarrhea and shedding were compared using the Fisher’s exact test. Statistical significance was assessed at P<0.05.
3.4 RESULTS
3.4.1 The HuNoV-HS66 caused intestinal lesions in the jejunum of a Gn calf
Intestinal tissue sections of two HuNoV-HS66-inoculated Gn calves and two mock-inoculated calves euthanized at PID 3 were analyzed. No lesions were observed in
203 the intestinal tissues of the mock-inoculated calf (Figure 3.1 A and B). Tissue sections of one of the two HuNoV-HS66-inoculated calves showed only mild atrophic enteritis with mild to moderate epithelial vacuolization (data not shown). The other calf had more severe loss of epithelial cells in most jejunal villi with moderate to severe diffuse atrophic enteritis and moderate to severe diffuse villous atrophy in the jejunum (Figure 3.2 A), but with milder lesions in the ileum and no lesions in the colon. Mild to moderate proliferation of crypt cells, increased numbers of MNC and a few necrotic cells were observed in the lamina propria of the same HuNoV-HS66-infected calf (Figure 3.2 B), compared to the mock-inoculated calf (Figure 3.1 A and B). No microscopic lesions were observed in the lungs, spleen, kidneys or liver of the 2 HuNoV-HS66-inoculated or the 2 mock-inoculated calves (data not shown).
3.4.2 Viral antigen was detected by immunohistochemistry (IHC) in the
jejunum of a HuNoV-HS66-inoculated calf
No positive cells were present in any tissues of the mock-inoculated calves (Fig
3A). Viral capsid antigen, detected as IHC positive cells (stained red), was present in enterocytes of the jejunum, and in a small number of enterocytes in the ileum of one
HuNoV-HS66 inoculated calf. Positive signals were also detected in macrophage-like cells present in the lamina propria (Figure 3.3 B).
3.4.3 HuNoV-HS66 caused diarrhea, viral shedding and viremia in Gn calves
After inoculation with HuNoV-HS66, 5 of 5 (100%) of the inoculated calves developed diarrhea and shed virus as detected by RT-PCR and microwell hybridization
204 assay (Table 1). Diarrhea was detected from PID 2-6 and viral shedding was detected in
the fecal samples of the calves by RT-PCR and microwell hybridization (PID 1-6) and
Ag-ELISA (PID 2-5), with a mean duration of shedding of 3 days by RT-PCR, 4 days by
microwell hybridization and 2 days by ELISA. One of 2 calves euthanized at PID 3 had
viral RNA detectable in the IC and one of 5 HuNoV-HS66-inoculated calves (20%) had
detectable viral RNA in the serum by RT-PCR (data not shown).
3.4.4 HuNoV-HS66 elicited 67% seroconversion and 33% coproconversion
rates in Gn calves
Low IgM, IgA and IgG antibody (Ab) titers were detected by
immunocytochemistry after inoculation with HuNoV-HS66 (geometrical mean titer;
GMT=20-80) (Figure 3.4). The IgM Ab was initially detected in serum at PID 4 (data not
shown) and peaked at PID 14. The IgA Ab was initially detected in serum at PID 7 and
peaked at PID 21 and 28 (GMT=63). Significantly elevated IgG Ab titers in serum were
first detected at PID 21 (GMT=40) and peaked at PID 28 (GMT=50). Two of 3 (67%)
HuNoV-HS66-inoculated calves euthanized at PID 28 seroconverted by PID 21-28 with
both IgA (titers from 20 to 320) and IgG antibodies (titers from 40 to 160). Low IgA Ab titers (GMT=20-40) were also detected in the IC of 2 of the 3 calves inoculated with
HuNoV-HS66 and euthanized at PID 28, and all 3 calves had IgG antibodies in their IC
although one of them did not have serum antibodies (data not shown). No HuNoV-HS66-
specific antibody responses were detected in the serum or IC of the control calves.
205 3.4.5 HuNoV-HS66 induced higher numbers of IgA and IgG ASC locally
compared to systemically
The numbers of IgA and IgG HS66-specific ASC detected by ELISPOT on PID
28 are shown in Figure 3.4. Low numbers of ASC were elicited in the HuNoV-HS66-
inoculated calves and no ASC were detected in the control calves. Low to moderate
numbers of IgA ASC were detected locally in the intestine (27 ASC per 5 x 105 MNC)
and MLN (11 ASC per 5 x 105 MNC) and systemically in spleen (17 ASC per 5 x 105
MNC) and blood (10 ASC per 5 x 105 MNC). Higher numbers of IgG ASC were elicited
in the intestine (77 ASC per 5 x 105 MNC) and also in spleen (21 ASC per 5 x 105
MNC), compared to IgA ASC.
3.4.6 HuNoV-HS66 induced a significant early peak (PID 2) of IFN-γ in
serum of Gn calves
The cytokine ELISA results are depicted in Figure 3.5. Overall, the HuNoV- inoculated Gn calves developed higher, or significantly higher, pro-inflammatory (TNF-
α), Th1 (IL-12 and IFN-γ), Th2/T-reg (IL-4 and IL-10) cytokines than control calves. The pro-inflammatory cytokine, TNF-α and Th1 cytokine IFN-γ both peaked early in infection at PID 2 (80 and 22 pg/ml, respectively) and again as a lower peak at PID 7 and
10 (42 and 54; 26 and 27 pg/ml, respectively), compared to controls. The Th1 cytokine
IL-12 was initially detected at PID 4 and peaked at PID 10 (174 pg/ml). Both IFN-γ and
TNF-α early serum peaks in the HuNoV-HS66-inoculated calves coincided with the onset of diarrhea (PID 2-6), viral shedding (PID 1-6) and viremia (PID 2).
206 The Th2 cytokine (IL-4) was detected only in serum of the HS66-inoculated
calves and peaked, significantly, at PID 4 (96 pg/ml), when compared to controls. The
Th2/T-reg cytokine (IL-10) was detected constitutively in serum of both HuNoV-HS66-
and mock-inoculated calves from PID 0-28, but with significantly higher levels detected
at PID 2, 4, and 7 in the HuNoV-HS66-inoculated calves when compared to controls. The
peak in the anti-inflammatory cytokine IL-10 concentration at PID 4 occurred shortly
after the early peak of the pro-inflammatory cytokine TNF-α and IFN-γ at PID 2.
3.4.7 HuNoV-HS66 induced significant early peaks of IL-12 (PID 2) and IFN-
γ (PID 4) in fecal samples of Gn calves
The concentrations of pro-inflammatory (TNF-α), Th1 (IL-12 and IFN-γ), and
Th2/T-reg (IL-4 and IL-10) cytokines were generally higher in the fecal samples of the
HS66-inoculated calves than in the control calves at all PID tested (Figure 3.6), but usually lower than the concentration of the corresponding cytokines in serum (Figure
3.5). In feces of the HuNoV-HS66-inoculated calves, in comparison to the serum cytokine profiles and kinetics, we found a similar pattern of TNF-α secretion with the highest peaks detected at PID 2 and PID 6 (16-14 pg/ml); corresponding to the viral shedding period (PID 1-6). However later (PID 21), increased TNF-α concentrations were only detected in feces. For Th1 cytokines, a significantly increased IL-12 peak (88 pg/ml) was detected earlier in infection (PID 2), compared to the later peak in serum (PID
10). In feces, increased concentrations of IFN-γ were initially detected at PID 1, and were significantly increased above the controls at PID 4, whereas the peak of IFN-γ in serum was at PID 2. For Th2/T-reg cytokines, a significantly increased peak of IL-4 in the fecal
207 samples was observed earlier (PID 1) than that of serum (PID 4); similarly the highest IL-
10 concentrations were detected early in the fecal samples (PID 1, significantly at PID 2),
compared to the serum peak concentrations (PID 4) (Figures 3.5 and 3.6).
The cytokine concentrations were measured in the IC of calves, after euthanasia at
PID 3 (n=4) and 28 (n=5). All cytokine concentrations tested were higher in the IC of the
HuNoV-HS66-inoculated calves than in the IC of control calves, but because of the small number of animals in the study and because of variability among animals and possible
degradation of some cytokines in IC, no statistical analysis was done (data not shown).
The highest mean concentrations of TNF-α and IFN-γ were detected in the IC of the
HuNoV-HS66-inoculated calves euthanized at PID 28 (164 and 26 pg/ml, respectively), compared to PID 3 (33 and 9 pg/ml, respectively). The IL-12 and IL-4 were each detected in similar concentrations at both PID 3 and 28. Only the IL-10 was detected at the highest mean concentration (840 pg/ml) early at PID 3.
3.4.8 HuNoV-HS66 induced in Gn calves high numbers of IFN-γ CSC both
locally (MLN) and systemically (spleen), high numbers of pro-inflammatory
(TNF-α) systemically (spleen and blood) and high numbers of Th2/T-reg (IL-
10) both locally (intestine) and systemically (spleen)
The numbers of CSC were higher both locally (intestine, MLN) and systemically
(spleen and blood) in the HuNoV-HS66-inoculated calves than in control calves (Figure
3.7). The pro-inflammatory (TNF-α) CSC numbers were highest systemically in spleen and blood (125 and 75 CSC/ 5 x 105 MNC, respectively) and lowest in intestine and
MLN (21 to 27 CSC/ 5 x 105 MNC) of the HuNoV-HS66-inoculated calves. Similarly,
208 the Th1 IL-12 CSC numbers were highest overall in the spleen (78 CSC/ 5 x 105 MNC)
and lowest in the MLN (18 CSC/ 5 x 105 MNC). In comparison, the highest IFN-γ CSC
numbers were detected in spleen (183 CSC/ 5 x 105 MNC), MLN (188 CSC/ 5 x 105
MNC) and intestine (130 CSC/ 5 x 105 MNC) with the lowest numbers in blood (42 CSC/
5 x 105 MNC) of the HuNoV-HS66-inoculated calves compared to controls. Although the
Th2 (IL-4) CSC were detected in the lowest numbers, they were highest in the intestine
compared to other tissues of the HuNoV-HS66-inoculated calves. The numbers of Th2/T- reg (IL-10) CSC both locally (intestine) and systemically (spleen) in the HS66-inoculated calves were the highest compared to the other cytokines.
3.5 DISCUSSION
Animal models are important to study the pathogenesis and immune responses to enteric caliciviruses. In this study, we assessed diarrhea and determined the enteropathogenicity, viral shedding, viremia and seroconversion of Gn calves experimentally infected with HuNoV-HS66 strain. We also evaluated the antibody titers in serum and IC and the ASC numbers in intestine, spleen and blood. The pro- inflammatory (TNF-α), Th1 (IFN-γ and IL-12), Th2 (IL-4), and Th2/T-reg (IL-10) cytokine profiles were also determined by ELISA in the serum, fecal samples and IC of
Gn calves. The CSC responses in intestine, spleen and blood were also determined by
ELISPOT in the Gn calves inoculated with the GII.4 HuNoV-HS66 strain or with mock
(controls) at PID 28.
We previously described the detailed pathogenesis of the HuNoV-HS66 strain in
gnotobiotic pigs (10). In this study, we present new evidence that the Gn calf also
209 constitutes an alternative experimental animal to study HuNoV pathogenesis and host
immune responses. Importantly, the occurrence of GIII NoVs in cattle also permits
comparative studies of host-specific versus NoV adoptive host strains. In the present
study, severe loss of epithelial cells in the villi, moderate to severe diffuse atrophic
enteritis and moderate to severe diffuse villous atrophy were detected mainly in the jejunum of a HuNoV-HS66-inoculated calf at PID 3. Increased MNC numbers and a few necrotic cells were observed in the lamina propria of this infected calf. In comparison,
experimental infection of Gn calves with a GIII NoV strain (NA-2) also showed more severe lesions in the jejunum of the small intestine, hypercellularity and the presence of
MNC and necrotic cells in the lamina propria (19). The Gn calves experimentally infected with an unrelated BEC (unassigned genogroup), the NB strain also had intestinal lesions that were more severe in the duodenum and jejunum and with milder lesions in
the ileum (44). We further detected HS66 capsid antigens in enterocytes of the jejunum,
and in smaller numbers in the ileum, with positive signals also being detected in
macrophage-like cells in the lamina propria. No positive cells were present in any tissues
of the mock-inoculated calves. In Gn pigs, only mild pathological changes were observed
in the intestine after infection with HuNoV-HS66 strain. Those changes included
multifocal villous atrophy, virus enterocytes with low columnar morphology and subtle
edema of the lamina propria of the duodenum; no changes were observed in the jejunum
and ileum. Viral antigen was distributed in a patchy manner in the villi of the duodenum and jejunum, and only in a few cells of the ileum. Furthermore, all the HuNoV-HS66- infected pigs showed a higher number of apoptotic cells compared to mock-inoculated pigs (10). The target cells for NoV replication in humans also appear to be the villous
210 enterocytes of the proximal small intestine. Biopsies of the jejunum of adult volunteers
who were infected with the GI NoV, NV or GII, Hawaii virus showed broadening and
blunting of the proximal intestinal villi with MNC infiltration and cytoplasmic
vacuolization (1).
Viral shedding evaluated by RT-PCR, hybridization and ELISA was also detected
in fecal samples from all infected calves with a mean duration of shedding of 4 days by
the hybridization assay. Both calves euthanized at PID 3 shed virus in feces from PID 1-
3, but only the IC of the calf with intestinal lesions was positive for viral RNA. Diarrhea
was observed in 5/5 (100%) of the infected calves, and viremia was detected in the serum
of one calf 1/5 (20%) at PID 2. Sixty-seven percent of the HS66-inoculated calves
seroconverted with low IgA and IgG antibody titers. Low IgA antibody titers were
detected in the IC of 2 (67%), and low IgG in the IC of all 3 calves euthanized at PID 28.
These results are very similar to those of HuNoV-HS66-infected Gn pigs (M. Souza, S.
M. Cheetham, M. S. P. Azevedo, V. Costantinini, and L. J. Saif, submitted for
publication) in which 91% of the pigs inoculated with HuNoV HS66 shed virus and 65%
of the pigs euthanized at PID 21 or 28 seroconverted to HuNoV HS66 with either IgA or
IgG antibodies, or with both.
Experimental Norwalk virus (NV) infection of non-human primates showed that
common marmosets and cotton top tamarins shed virus for only 2 days and only one of
four NV-inoculated rhesus macaques, that shed NV for a long period in the feces,
developed serum NV-specific IgM and IgG antibody responses. No IgA NV-specific antibodies were detected in the plasma or saliva of this animal. Clinical signs were not observed in any of the animals (43). Data on the role of secretory IgA (sIgA) in HuNoV
211 infections are conflicting; however, a correlation between salivary sIgA and protection
against NV infection has been recently established, although not all susceptible
individuals that did not become infected had strong salivary sIgA responses (28).
The numbers of ASC induced systemically (spleen and blood) of HuNoV-HS66-
infected calves were generally low, and similar to those of the Gn pigs infected with the
HuNoV-HS66 strain, reflecting localized intestinal viral replication and transient viremia
detectable only in one of the 5 HuNoV-HS66-inoculated calves. However, higher
numbers of IgG ASC (77 ASC/ 5 x 105 MNC) were detected in the intestine of the HS66- inoculated calves at PID 28 when compared to low numbers in the HuNoV-HS66- inoculated Gn pigs (4 ASC/ 5 x 105 MNC) (M. Souza, S. M. Cheetham, M. S. P.
Azevedo, V. Costantini, and L. J. Saif, submitted for publication). This higher number of
IgG ASC in the intestine of HS66-infected Gn calves corroborates the more extensive intestinal lesions observed in the calves when compared to the similarly inoculated Gn pigs (10).
The seroconversion rates found in this study (67%) were also very similar to those found in the Gn pig study (65%), but higher serum IgA and IgG antibody titers (GMT=50 to 63) were detected in comparison to those in the Gn pigs (GMT=18 and 14,
respectively). Low IgA and IgG antibody titers (GMT = 20-40 and 20-80, respectively)
were also detected in 67%, and 100%, respectively of the IC of HS66- infected calves at
PID 28. Similarly in the Gn pigs, 64% of the IC from the HuNoV-HS66-infected pigs had
IgA or IgG antibodies (M. Souza, S. M. Cheetham, M. S. P. Azevedo, V. Costantini, and
L. J. Saif, submitted for publication).
212 In humans, seroconversion rates vary (50-90%) (33), local and secretory immune
responses to HuNoV infections have been poorly documented (6, 37), and the association
between local jejunal antibody titers and resistance to NV infection has not been
established in adult volunteers orally exposed to this virus (17). Because most adult
volunteers have pre-existing antibodies to HuNoVs upon challenge, paired serum
samples are needed for interpretation of the antibody responses to viral challenge.
Primary immune responses cannot be easily assessed because adult humans are frequently exposed to these viruses during their lifetime. Thus, the Gn pig, together with
the Gn calf model, may provide important information on the primary immune responses
to HuNoV, and especially the local intestinal immune responses.
There is little information on the cytokine responses to HuNoVs. The
concentrations of the pro-inflammatory cytokine (TNF-α) increased in the serum of
HuNoV-HS66-inoculated calves at PID 2, 7 and 10, when compared to control calves, coinciding with the peaks of IFN-γ and the presence of diarrhea at PID 2-6. In the fecal samples, increased levels of TNF-α, although not significantly higher than controls, were observed at PID 1, 2, 6 and later at PID 21, and higher levels were detected in the IC of
HuNoV-HS66-inoculated calves at PID 3, compared to controls. Higher numbers of
TNF-α CSC in spleen and of IFN-γ CSC in MLN, intestine and spleen of HuNoV-HS66- inoculated calves were also detected. Significant increases in IFN-γ secretion were detected in the serum of volunteers two days after Snow Mountain virus (SMV) challenge (27). In our study, we also detected increased IFN-γ concentrations in the serum and fecal samples of HuNoV-HS66-inoculated Gn calves at PID 2 and 4, respectively, corroborating the early increase of IFN-γ also seen in the serum of HuNoV-
213 HS66-infected pigs (M. Souza, S. M. Cheetham, M. S. P. Azevedo, V. Costantinini, and
L. J. Saif, submitted for publication), and confirming the early induction of Th1 (IFN-γ)
responses during viral infection (40). This early IFN-γ peak (PID 2) in serum and in fecal samples (PID 4) is likely due to an early innate immune response to viral replication (30),
whereas the later peaks (PID 7 and PID 10 in serum), were most likely elicited in response to the increased levels of the Th1-inducer (IL-12) at PID 2, 4, 6 and 10 in the
feces and at PID 4, 7 and 10 in serum (12, 22, 47). The early increases of IL-12 in the serum and fecal samples (starting at PID1) may represent the early innate responses of macrophages and dendritic cells to NoV infection (8).
A similar synergy between IFN-γ and TNF-α has been reported during
Toxoplasma infections in mice (26), and increased levels of both cytokines were also detected in the serum of Gn pigs infected with virulent human rotavirus (HRV) (4). Low to moderate levels of IL-6, another pro-inflammatory cytokine, were also detected in
SMV-challenged humans by Lindesmith et al. (27), and low to moderate levels of this same cytokine were also detected in the serum of Gn pigs infected with HuNoV-HS66 strain at PID 4 (M. Souza, S. M. Cheetham, M. S. P. Azevedo, V. Costantinini, and L. J.
Saif, submitted for publication).
The induction of IFN-γ and TNF-α or their CSC locally (fecal samples, IC, intestine and MLN), and systemically (serum and spleen) may reflect the host response to virus replication and intestinal pathology observed resulting in gut inflammation. The
levels of the pro-inflammatory cytokine IL-6 were significantly elevated at PID 1 and 3
in HRV-infected pigs reflecting the high virus load during replication in the gut and the
extensive intestinal pathology induced (4).
214 The Th2/T-reg (IL-4 and IL-10) cytokines were also elicited early in infection both locally (fecal samples and IC) and systemically (serum) in HuNoV-HS66-infected calves. Early significant increases in IL-4 concentrations were detected in the fecal samples at PID 1 (13-fold compared to controls) and only later at PID 7 (10-fold compared to controls) in the serum of the HuNoV-HS66-inoculated calves. Higher levels, although not significantly higher than controls, were also detected in the fecal samples at
PID 2, 4 and 6, and in the serum at PID 2 and 4. Significantly higher levels of IL-4 were also detected in HuNoV-inoculated Gn pigs at PID 2, 4, 6 and 8 when compared to controls (M. Souza, S. M. Cheetham, M. S. P. Azevedo, V. Costantinini, and L. J. Saif, submitted for publication). Higher (although not significantly higher) peaks of the
Th2/T-reg (IL-10) cytokine were also detected in the fecal samples of HuNoV-HS66- inoculated Gn calves at PID 1, and 10 and in the IC at PID 3, when compared to control calves. Significantly higher levels (up to 4-fold) were also detected in the serum of HS66- inoculated calves at PID 2, 4 and 7, when compared to controls.
Higher numbers of both IL-4 and IL-10 CSC were also detected in the HuNoV-
HS66-inoculated calves locally in intestine, and systemically for IL-10 (spleen) at PID
28. However, the numbers of IL-10 CSC in the intestine and spleen were dramatically higher than those of IL-4 (154 and 251; 15 and 8, respectively). When adult volunteers were challenged with SMV, no significant changes were detected between pre and post- challenge concentrations of IL-10 in their serum samples, and significant changes were not detected between pre-challenge and post-challenge PBMC secretion of IL-4 or IL-10 after vitro stimulation with SMV (27). However, significantly higher levels of IL-5, another Th2 cytokine, were observed, confirming that the GII.2 HuNoV induced a Th1
215 immune response, but not exclusively. The differences in the IL-4 and IL-10 serum responses in calves versus humans may reflect differences due to previous NoV exposure history in adult humans versus seronegative neonatal calves, requiring similar cytokine studies in NoV infected seronegative infants to clarify these differences.
The IL-10 is a regulatory cytokine in pigs and humans (15, 34, 36), with a role in the control of inflammation (15). Human IL-10 inhibits IFN-γ and TNF-α synthesis, and during parasitic infections in cattle, an increase in IL-10 with a decrease in IFN-γ synthesis was also detected (46). Increased levels of IL-10 were detected early in the fecal samples (PID 1) and again later (PID 10), with higher levels also observed in the IC
(PID3) of the HuNoV-HS66-inoculated calves. Significantly higher IL-10 concentrations at PID 4 (650 pg/ml) were detected in the serum of the HuNoV-HS66-inoculated calves that coincided with decreased IFN-γ and TNF-α concentrations that increased again at
PID 7 after the concentrations of IL-10 diminished (369 pg/ml).
Significantly enhanced levels of IL-10 were also detected in the serum of
HuNoV-HS66-infected Gn pigs at similar PIDs to those of the Gn calves; however, at much lower concentrations (maximum of 15 pg/ml) compared to those in the serum of
Gn calves (maximum of 650 pg/ml). The Th2 (IL-4) and Th2/T-reg (IL-10) cytokines were significantly elevated early (PID 2 or 4, 6, 8), and were detected at moderate to low levels at most PIDs in the serum of HuNoV-HS66-infected pigs (M. Souza, S. M.
Cheetham, M. S. P. Azevedo, V. Costantinini, and L. J. Saif, submitted for publication).
At PID 28, higher numbers of IL-10 CSC were also detected in the intestine and spleen of
HS66-inoculated calves, compared to control calves. These data corroborate the regulatory and anti-inflammatory actions of IL-10, both locally and systemically, during
216 viral infections. We further demonstrate that the HuNoV-HS66 strain induced a higher
level of pathology and inflammation (TNF-α) in Gn calves when compared to Gn pigs
(M. Souza, S. M. Cheetham, M. S. P. Azevedo, V. Costantinini, and L. J. Saif, submitted
for publication).
In conclusion, the HuNoV-HS66 strain induced low levels of antibodies and low to
moderate numbers of ASC both systemically and in the intestine with 67%
seroconversion in calves. The HuNoV-HS66 induced, both locally (feces) and
systemically (serum) low to moderate Th1 (IFN-γ and IL-12) and Th2 (IL-4) responses, and higher Th2/T-reg (IL-10) systemically (serum), corroborating our previous results of the induction of both type of responses in Gn pigs by the same HuNoV strain. The early
local and systemic peaks of the pro-inflammatory TNF-α and its increased concentrations
in the IC of the HuNoV-HS66-inoculated calves during viral shedding and diarrhea, in
concert with the intestinal lesions shown by histopathology, suggest that the HuNoV-
HS66 strain induced more pronounced intestinal lesions in Gn calves compared to the Gn
pigs (10). This may reflect the wider prevalence of NoV (GIII) observed in association
with diarrhea in young calves reflecting their pronounced susceptibility to NoV infection
(44). However, both Gn animals appear to be suitable models for the study of HuNoV
pathogenesis and host immune responses.
This study provides new information on the enteropathogenicity, antibody levels, cytokine secretion kinetics, both locally and systemically, in response to HuNoV-HS66 infection of Gn calves and supports the use of the Gn calves as an experimental animal model for GII HuNoV infection and disease.
217 3.6 ACKNOWLEDGMENTS
We thank Dr. Mary Estes (Baylor College of Medicine) for kindly providing the
NS14 monoclonal antibody. We also thank Dr. J Hanson, Mr. R. McCormick and Mr.
Greg Meyers for animal care and Dr. J. H. Hughes, who kindly provided the original
NoV GII.4 human fecal sample.
Salaries and research support were provided by state and federal funds appropriated to the Ohio State University. This work was supported by a grant from the
National Institute of Allergy and Infectious Diseases, National Institutes of Health, grant
No. R01-AI49742 to the corresponding author (L. J. Saif). Menira Souza was a fellow of
Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Brasilia,
Brazil, from July-2002 to July 2005.
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223 Inoculum Virus Diarrheab Diarrhea Viremiad Seroconversion e Coproconversion ICf # calves sheddinga (%) (%) (%) (%) mean mean mean cumulative days days scorec (range) (range) (range) HS66 3Ag (1-6) 5/5 (100%)A 3 (2-6) 10A (2-14) 1/5 (20%)A 2/3 (67%)A 1/3 (33%)A (n=5)
Control 0B 1/5 (20%)B 0.2 2B (1-4) 0/5 (0%)B 0/2 (0%)B 0/2 (0%)B
224 (n=4)
aMean number of days and range of shedding determined by RT-PCR bDiarrhea present if fecal swab scores were ≥2 after inoculation. cRepresents the sum of daily rectal swab scores from PID 1-6 of each calf divided by the number of calves in that group dViremia was determined by RT-PCR on PID 2 efSeroconversion and coproconversion were determined by immunocytochemistry (HS66 recombinant baculovirus infected Sf9 cell-staining assay) at PID 21 and/or 28 gValues in the same column with different superscript letters (A or B) differ significantly (p<0.05)
Table 3.1. Diarrhea, fecal virus shedding and viremia detected by RT-PCR and seroconversion in Gn calves inoculated with
either HuNoV-HS66 or mock-inoculated controls.
A B
Figure 3.1. Histological examination of the intestine of a mock-inoculated calf. A. Jejunum at post-inoculation day 3. No lesions were detected (original magnification x 75). B. Same as A at a higher magnification ( x 150). Hematoxylin
A B
Figure 3.2. Histological examination of the intestine of a HuNoV-HS66-inoculated calf. A. Jejunum at PID 3. Moderate to severe diffuse villous atrophic enteritis (30 to 50% loss of villi), mild to moderate proliferation of crypt cells (closed arrows) and increase of cell populations in the lamina propria were observed (arrow head) (original magnification x 75). B. Jejunum at PID 3 (x 200). Severe loss of epithelial cells (closed arrows) was observed in most villi of the jejunum. Mononuclear cells with dense nuclei (arrow head) were mildly increased in the lamina propria. Exfoliated necrotic cells and debris (open arrow) were occasionally observed in the intestinal lumen. Hematoxylin and eosin stain.
225
A B
Figure 3.3. Imunohistochemistry for detection of GII HuNoV capsid antigens. A. Jejunum of mock-inoculated calf at PID 3. No positive cells were observed (original magnification x 150). B. Jejunum of HuNoV-HS66-inoculated calf at PID 3. Viral capsid antigens (stained red) were observed in the cytoplasm of a few intestinal epithelial cells attached to the villi or exfoliated enterocytes (arrow heads) and occasionally in the cytoplasm of crypt epithelial cells (closed arrows) (original magnification x 200). IHC; 3, 3’-diaminobenzidine (DAB); Mayer’s hematoxylin counterstain.
226 Serum IgM Ab Serum IgA Ab 200 Serum IgG Ab 200 200 150 150 150 100 100 100
GMT *
GMT * 50 GMT 50 50 * 0 0 0 7142128 7 142128 7142128 PID PID PID
IgA ASC at PID 28 IgG ASC at PID 28
MNC 150
150 5 MNC 227 5 100 100 50 50 0 0 Intestine MLN Spleen Blood Intestine MLN Spleen Blood 5ASC/ X 10 ASC/ 5ASC/ X 10
Figure 3.4. Isotype-specific (IgM, IgA and IgG) antibody titers and antibody secreting cell mean numbers in intestine, MLN, spleen and
PBMC of gnotobiotic calves inoculated with HS66 or controls. Symbols: ■ , HS66; □ controls.* significantly higher than controls (P<0.05) Cytokine in Serum Th1 IFN-γ in serum IL-12 in serum 50 400 40 300 30 * 200 20 pg/ml 100 pg/ml 10 0 0 024710142128 024710142128 PID PID
Pro-inflammatory
TNF-α in serum 200
228 150 100
pg/ml 50 0 0 2 4 7 10 14 21 28 PID Th2/T-reg IL-4 in serum IL-10 in serum 200 * 1000 * 150 800 600 * 100 * * 400
50 pg/ml
pg/ml 200 0 0 0 2 4 7 10 14 21 28 0 2 4 7 10 14 21 28 PID PID
Figure 3.5. Th1 (IL-12 and IFN-γ), pro-inflammatory (TNF-α), Th2 (IL-4) and Th2/T-reg (IL-10) cytokine concentrations in serum of gnotobiotic
calves inoculated with HuNoV-HS66 or controls. Symbols: HuNoV-HS66; , controls. * significantly higher than controls (P<0.05). Cytokine in Feces
IL-12 in fecal samples IFN-γ in fecal samples 200 Th1 30 25 150 * 20 * 100 15 pg/ml 50 pg/ml 10 5 0 0 0 1 2 4 6 10 14 21 01246101421 PID PID Pro-inflammatory
TNF-α in fecal samples 30 25 20 15 229 10 pg/ml 5 0 0 1 2 4 6 10 14 21 PID
IL-4 in fecal samples Th2/T-reg 150 IL-10 in fecal samples 125 75 100 50 75 * 50 *
pg/ml 25 25 pg/ml 0 0 01246101421 01246101421 PID PID
Figure 3.6. Th1 (IL-12 and IFN-γ), pro-inflammatory (TNF-α) and Th2/T-reg (IL-10) cytokine concentrations in fecal samples
of gnotobiotic calves inoculated with HuNoV-HS66 or Mock contr Symbols: HS66; controls.
* significantly higher than controls (P<0.05). , controls.
γ IFN- IL-10 Intestine MLN Spleen Blood Intestine MLN Spleen Blood
0
0
MNC
300 200 10 100 x 5 CSC/
500 400 300 200 100 5 5 MNC 10 x 5 CSC/ 5 5 g Th1 ) and Th2/T-reg (IL-4 and IL-10) cytokine secreting cell mean numbers in ) and Th2/T-reg (IL-4 IL-10) cytokine α α Th2/T-re Pro-inflammatory CSC at PID 28 TNF- Intestine MLN Spleen Blood IL-12
), pro-inflammatory (TNF-
IL-4 γ 0
MNC 300 200 10 100 x 5 CSC/ 5 5 Intestine MLN Spleen Blood Intestine MLN Spleen Blood 0
5 0 300 200 100 MNC
CSC/ 5 x 10 x 5 CSC/ 25 20 15 10
MNC 10 x 5 CSC/ 5 5 5 5 intestine, MLN, spleen and blood of gnotobiotic calves inoculated with HuNoV-HS66 or controls. Symbols: , HS66; Figure 3.8. Th1 (IL-12 and IFN-
230 CHAPTER 4
A HUMAN NOROVIRUS-LIKE PARTICLE VACCINE ADJUVANTED WITH
ISCOM OR MLT INDUCES CYTOKINE AND ANTIBODY RESPONSES AND
PROTECTION TO HOMOLOGOUS VIRUS IN A GNOTOBIOTIC PIG
DISEASE MODEL
4.1 SUMMARY
Human norovirus (HuNoV) affects people of all ages worldwide causing diarrheal disease. Safe and efficacious vaccines are needed to prevent HuNoV infection, especially in highly susceptible populations. We used the gnotobiotic (Gn) pig model to evaluate antibody and cytokine responses, both locally and systemically, to an oral/intranasal (IN)
HuNoV GII.4 (HS66 strain) virus-like particle (VLP) vaccine, with the mucosal adjuvants, immunostimulating complexes (ISCOM) (group 1) and mutant E. coli heat labile toxin (R192G) (group 2), compared to each adjuvant alone (controls, group 3). We also evaluated protection against homologous (HuNoV-HS66) viral challenge. All pigs challenged with HuNoV-HS66 had A+/H+ histo-blood group phenotype. The protection rate against virus shedding was 100% for both vaccinated groups and against diarrhea was 75% in the VLP+ISCOM group and 100% in the VLP+mLT group; however, only
57% of the controls shed virus post-challenge. Seroconversion was detected in 100% of
231 the vaccinated pigs and coproconversion occurred in 100% of the VLP+ISCOM and in
75% of the VLP+mLT vaccinated pigs. Local (intestine) and systemic (spleen and blood)
antibody secreting cell (ASC) responses were evaluated. The VLP+ISCOM vaccine
elicited higher IgM, IgA and IgG intestinal ASC responses compared to VLP+mLT, both
pre- and post-challenge. The Th1 (IL-12 and IFN-γ), pro-inflammatory (IL-6), Th2 (IL-4 and IL-13) and Th2/T-reg (IL-10) cytokine concentrations were measured in serum and intestinal contents (IC) of all pigs. Although both vaccine regimens elicited increased levels of IFN-γ in serum, and higher numbers of IL-12 and IFN-γ CSC in spleen post- challenge. VLP+mLT induced higher Th1 and Th2 CSC numbers pre-challenge and dramatically higher post-challenge levels of IFN-γ in the IC of the vaccinated pigs compared to the VLP+ISCOM and control groups. Thus, both vaccine regimens induced high seroconversion, coproconversion and protection against viral shedding and diarrhea.
Use of the Gn pig challenge model to assess protective immunity to HuNoV GII.4 VLP
vaccines has provided important new information on both the immunity and protection
induced confirming the potential efficacy of NoV VLP vaccines for humans (325).
4.2 INTRODUCTION
The HuNoVs constitute the major cause of nonbacterial, epidemic gastroenteritis
worldwide, and in the US alone they are responsible for 23 million cases of
gastroenteritis and 50,000 hospitalizations yearly (24). Norovirus disease is usually
characterized by self-limited gastroenteritis and/or vomiting with symptoms lasting
approximately 12-48 hrs; however, it accounts for a significant disease burden associated
with numerous outbreaks in settings such as nursing homes (2), hospitals (19), cruise
232 ships (1, 18) and the military (3). Asymptomatic infections by HuNoV are also very
common which may be important for HuNoV person-to-person transmission (23).
Recombinant NoV capsids expressed as VLPs constitute a safe alternative for the
development of HuNoV vaccines (6). These particles are antigenically similar to the
native virion, can be produced at high yields using the baculovirus system, and are stable
at low pH (27), making them suitable for oral administration and mimicking the natural
route of HuNoV infection (11).
Different strategies, including Venezuelan equine encephalitis (VEE) replicon
system, transgenic plants, yeast and the recombinant baculovirus system, have been used
to produce HuNoV VLP. Their efficacy has been evaluated in mice and in humans (7,
21). The GI Norwalk-virus (NV) VLPs were administered IN to BALB/c mice in the
presence or absence of the mucosal adjuvant mutant Escherichia coli heat-labile toxin
R192G (mLT). Low doses (10-23 μg) of NV VLPs were immunogenic, and higher doses
(200 μg) induced specific serum IgG, fecal and vaginal antibody responses. The use of
mLT enhanced the immune responses (13). The oral administration of 250 μg, or more of
NV VLPs to human volunteers elicited 90% seroconversion and increased numbers of
NV-specific IgA antibody-secreting cells (ASC) in peripheral blood mononuclear cells
(PBMCs), with 30-40% of the volunteers developing specific mucosal IgA antibodies
(30).
The mLT (R192G) toxin has a single amino acid substititution in position 192 that
diminishes its toxicity while still retaining its adjuvanticity when administered orally or
IN (14, 22, 35). Immunostimulating complexes (ISCOM) also constitute an alternative as
both delivery system for antigens and as adjuvant. These cage-like structures are
233 composed of subunits built from the interaction of the surfactant saponins with lipid
particles (cholesterol and phospholipids) (31). Previous studies using the Gn pig model
showed that oral priming with attenuated human rotavirus (HRV) followed by 2 IN
booster doses of RV inner capsid proteins VP2/VP6 in 2/6 VLPs in combination with
mLT or ISCOM, induced protection rates against diarrhea and virus shedding similar to
those of the pigs that received the three-dose attenuated human rotavirus (AttHRV)
vaccine (17), and boosted antibody titers and ASC responses (17, 26, 35). This vaccine
regimen provided high protection rates against diarrhea after challenge with virulent
WaHRV and induced high virus neutralization and mucosal IgA antibody titers to HRV
in the intestinal contents of Gn pigs (12). Similar strategies using ISCOM or mLT
adjuvanted HuNoV VLP vaccines have not been tested in humans.
Immunity to HuNoV is complex, and is largely undefined due to the absence of
relevant NoV animal models of enteric disease and limitations associated with human
volunteer studies. Some of these studies that used paired pre- and post-challenge serum
samples from human volunteers may have confounding results due to unknown previous,
and sometimes multiple, exposures to HuNoV strains. The ABO histo-blood group type
and the secretor status have been identified as genetic factors that may influence
susceptibility to norovirus (NoV) infection and/or disease in humans (16). We have
recently demonstrated that pigs with the A+ and/or H+ phenotype had increased diarrhea and viral shedding rates compared to Gn pigs of non- A+ or H+ phenotypes infected with a GII.4 HuNoV strain (8, 9). However, GII.2 Snow Mountain virus (SMV) infection was not dependent on the histo-blood group or secretor status (21), and a more recent study showed that VLPs of the Bristol-like GII.4 strains, that have been implicated in various
234 outbreaks globally for the last 10 years, did not bind to the saliva of 78% of secretor-
positive individuals (20), regardless of their blood type, a binding pattern similar to that
of the Lordsdale-like (LV) GII.4 strains (15), raising questions about the role of these
antigens for certain HuNoV strains in binding to and entering into their target cells.
Local immunity to HuNoV is difficult to assess and few studies, either in mice or
in humans, have been done (7, 20, 21). The Gn pig constitutes a valuable model for the
study of systemic and local immunity to enteric pathogens due to its resemblance in gut physiology to humans and lack of pathogen exposure, allowing assessment of primary
immune responses. We have recently conducted a detailed study on the local and
systemic cytokine and antibody responses to a GII.4 HuNoV (HS66) strain in the Gn pig
model (M. Souza, S. M. Cheetham, M. S. P. Azevedo, V. Costantini, and L. J. Saif,
submitted for publication), and showed that this strain induced both Th1 and Th2 immune
responses locally and systemically.
In this study we evaluated the systemic and local immune responses of Gn pigs to
one oral followed by two IN booster doses of HuNoV GII.4 HS66 strain (HuNoV-HS66)
VLPs with mLT or ISCOM adjuvant, both prior to and after viral challenge with
homologous virus and the induction of protection.
4.3 MATERIALS AND METHODS
4.3.1 Recombinant HS66 VLPs
The HuNoV-HS66 strain VLPs were produced using the recombinant baculovirus
system, as previously described (9), with minor modifications. Briefly, a recombinant
baculovirus that contained the capsid gene sequence of the HuNoV-HS66 strain was
235 constructed (9) and, after a baculovirus stock was produced and plaque purified, the
VLPs were produced by infecting the Spodoptera frugiperda (Sf9) cells with the HS66
baculovirus stock at a multiplicity of infection (moi) of 8, incubated at 27ºC and
harvested at post-inoculation day (PID) 10. The supernatants were collected and
centrifuged at 3,000 X g for 30 min to pellet the cells and the assembled VLP were purified by CsCl density gradient ultracentrifugation, as previously described (9). Particle
integrity and reactivity were tested by immune-electron microscopy (IEM), antigen enzyme linked immunosorbent assay (ELISA) and Western blotting, and protein concentration was evaluated by the Bradford quantification method (Bio-Rad, Hercules,
CA) (9). The sterility of each VLP preparation was assessed by bacteriologic culture assays using blood agar plates and thioglycolate broth, at both room temperature (Rt) and at 37ºC, for two weeks. The endotoxin levels were measured by using the Limulus
amebocyte lysate assay (Association of Cape Cod, Woods, Hole, Mass) (17).
4.3.2 VLP-ISCOM vaccine
The HuNoV-HS66 VLPs were mixed with 2 M LiCl2 for 30 min at Rt and then
incubated overnight at -70ºC to increase their capacity to bind to the ISCOM matrix (17).
The LiCl-treated VLPs (positively charged by the Li++ ions) were mixed with ISCOM matrix (5 mg of ISCOM per 1 mg of HuNoV-HS66 VLPs) and then dialyzed in 0.09%
NaCl solution for 72 hrs, and the association of the HuNoV-HS66 VLPs with ISCOM
matrix was confirmed by IEM (12).
236 4.3.3 VLP-mLT vaccine
We used mLT containing a mutation (from arginine to glycine, R192G) at amino
acid position 192 of the protein, kindly provided by J. Clements (Tulane University
Medical Center, New Orleans, La.). The dose used in this study (5μg) was as previously
determined in Gn pig experiments in our lab using HRV VLPs (35). The HuNoV-HS66
VLPs were mixed with the mLT (5 µg of mLT per 250 mg of VLPs), previously diluted
in phosphate buffer saline (PBS) pH 7.2, immediately preceding their use.
4.3.4 HuNoV-HS66 virus challenge inoculum
A single aliquoted pool of the original human fecal sample identified as
NoV/GII/4/HS66/2001/US (HS66 strain) (9) was used for oral inoculation of Gn pigs
using a dose of approximately 5.4 x 106 genomic equivalents (GE)/ml. A uniform inoculum pool was prepared by diluting the HuNoV original inoculum 1:10 in minimal
essential medium (MEM) (Gibco, Invitrogen, Carlsbad, CA), further processing it by
vortexing, centrifugating at 3000 x g for 20 min and filtration through 0.8 μm followed
by 0.2 μm filters. A subset of the pigs were challenged with one oral dose (10 ml of the
1:10 dilution) of the pooled inoculum after being administered 8 ml of 100mM sodium
bicarbonate, to neutralize stomach acids.
4.3.5 Inoculation of the experimental pigs
Near-term pigs were derived by surgery and maintained in sterile isolator units as
previously described (25). The 30 Gn pigs used in this study were allocated into one of
three groups: (1) one oral and two IN immunizations with 250 µg of HuNoV-HS66-VLP 237 + 1,250 µg of ISCOM (n=8); (2) one oral and two IN immunizations of 250 µg of
HuNoV-HS66 VLP + 5 µg of mL (n=8); and the control group: (3) one oral and two IN immunizations with 1,250 µg of ISCOM matrix or 5 µg of mLT (n=14). The first inoculation of the Gn pigs was performed at 5 days post-derivation at post-inoculation day (PID) 0, and it was preceded by 8 ml of 100mM of sodium bicarbonate orally. The other two subsequent IN inoculations were at PID 10 and PID 21. Half of the Gn pigs in each group were euthanized at PID 28 (after receiving one oral and two IN doses of the respective vaccine) and the other half was challenged with the original HuNoV-HS66 inoculum at PID 28 and euthanized at PID 35/post-challenge day (PCD) 7.
4.3.6 A/H typing
The A/H phenotype of each pig, at 3 to 5 days of age, was determined by hemagglutination inhibition (HI) using buccal cells as previously described (8).
4.3.7 Assessment of diarrhea
Daily rectal swabs were collected and diarrhea scores were noted and recorded
(0=normal; 1=pasty; 2=semi-liquid; 3=watery) from PID 28-35 as described previously
(9). Samples with scores 2 and 3 were considered diarrheic. The diarrhea cumulative score of each pig represents the sum of daily rectal swab scores from PCD 1-6, and the mean cumulative score of each group is the sum of each pig’s diarrhea cumulative score divided by the number of pigs in that group.
238 4.3.8 Viral shedding by RT-PCR
Viral shedding was determined using rectal swab fluids and 1:20 dilutions of intestinal contents (IC) by RT-PCR, using the primer pair Mon 431/433 (29) targeting the
RdRp region of HuNoV GII, using the same conditions as previously described (9).
However unlike as previously described, samples that were inhibited in RT-PCR, as revealed by the use of an internal control (9), were re-tested after being re-extracted using the RNeasy Mini kit (Qiagen Inc, Valencia, CA). Negative controls (rectal swabs from mock inoculated pigs and RNAse-free water) for RNA extraction and RT-PCR were included in each assay. A microplate hybridization assay (33) was performed to confirm the product specificity using a probe specific for HuNoV-HS66 (9).
4.3.9 Viral shedding by antigen-ELISA
The antigen ELISA was performed as previously described by Cheetham et al.
(9). Samples were considered positive when the mean absorbance (450 nm) of the positive coating wells minus the mean absorbance of the negative coating wells was higher than the mean absorbance of the negative control wells plus 3 times the standard deviation.
4.3.10 Antibody detection by immunocytochemistry
An immunocytochemistry assay was performed to detect HS66-specific antibodies in the serum and IC of Gn pigs, as previously described (36). For this assay a recombinant baculovirus expressing HS66 capsid was used to infect Spodoptera frugiperda (Sf9) cells as the HuNoV antigen source and the recombinant baculovirus
239 infected cells or mock cells (infected with an unrelated recombinant baculovirus) were
subsequently fixed using 10% formalin in PBS. The antibody titer was defined as the
reciprocal of the highest serum dilution at which brown-stained cells representing NoV
antibody complexed to HuNoV-HS66 capsid antigen could be detected.
4.3.11 Isolation of mononuclear cells (MNC) for ELISPOT assays
Segments of the small intestine (jejunum and ileum), spleen, and blood were aseptically collected at euthanasia and processed for the isolation of MNC populations, as
previously described (32, 37). Single MNC suspensions from each tissue and blood were
prepared at concentrations of 5 x106 MNC and 5 x105 MNC/ml in complete medium prepared with Roswell Park Memorial Institute (RPMI)1640 (GIBCO) enriched with 8% fetal bovine serum, 20mM Hydroxyethyl-Piperazine Ethanesulfonic Acid (HEPES), 2mM
L-glutamine, 1mM sodium pyruvate, 0.1mM nonessential amino acids, 100 μg of
gentamicin/ml, 100 μg of ampicilin/ml, and 50 μg of 2-mercaptoethanol.
4.3.12 ELISPOT assay for detection of HuNoV-HS66-specific antibody-
secreting cells (ASC)
An ELISPOT for detection of isotype-specific (IgM, IgA and IgG) ASC was
conducted using previously published methods (10, 38). Briefly, Sf9-cell plates infected
with the HuNoV-HS66 capsid gene recombinant baculovirus and mock plates were
prepared and fixed as described in the antibody detection section and washed with
deionized water prior to use. Single MNC suspensions from each tissue were added to
duplicate wells (5 x105 and 5 x104 /well). Plates were then incubated at 37°C for 12 hrs in 240 5% CO2 and then washed three times with PBS buffer and incubated at 37°C for 2 hrs
with 100 μl/well of HRP-labeled antibody: goat anti-pig IgM (μ) (KPL) (0.25 μg/ml);
IgA (Serotec) (0.3 μg/ml); or IgG (KPL) (0.25 μg/ml). Plates were then washed three times in PBS buffer and developed with TMB (KPL Inc.) for 2 hrs at Rt. The number of
virus-specific ASC were determined by counting blue spots in the wells, using a light
microscope, and were reported as the number of virus-specific ASC per 5 x 105 MNC, after any background spots (< 3), evident on the mock plates, were subtracted.
4.3.13 ELISPOT assay for detection of HuNoV-HS66-specific cytokine-
secreting cells (CSC)
A cytokine ELISPOT for detection of pro-inflammatory (IL-6), Th1 (IL-12 and
IFN-γ), Th2 (IL-4) and Th2-T-reg (IL-10) CSC was performed as previously described
(5) with minor modifications. Before being added to Multiscreen-IP sterile 96-well plates
(Millipore, Bedford, MA) at concentrations of 5 x105 and 5 x104 /well, the cells were
stimulated with 50 μg/ml of CsCl-purified HuNoV-HS66 virus-like particles (VLPs) (9)
or 10 μg/ml of phytohemagglutinin (positive control) or RPMI (negative control). Plates
were then incubated at 37°C in 5% CO2 for 48 h. The numbers of CSC were counted
using an ImmunoSpot series 3A analyzer (Cellular Technology Ltd., Cleveland, OH) and
expressed as CSC per 5 x105 MNC. The HuNoV-HS66-specific CSC numbers were
computed after the numbers of CSC (< 4) in the controls (RPMI-stimulated cells) were
subtracted from the HS66 VLP-stimulated cells.
241 4.3.14 Cytokine concentrations in the serum and IC quantitated by cytokine
ELISA
Blood was collected from pigs at PID 0, 2, 7, 10, 21, 28, 30 and 35 and intestinal contents were collected at euthanasia (PID 28/PCD 0 and PID35/PCD7). Serum samples were processed and stored at -20° C (5). The IC samples were diluted 1:2 in MEM with a protease inhibitor cocktail to prevent cytokine degradation (4). The serum and IC were immediately frozen at –20°C until further testing. An ELISA test was performed to detect
IL6, IL-12, IFN-γ, IL-4 and IL-10 as previously described (5). In addition to those cytokines, an ELISA to detect the porcine Th2 cytokine, IL-13 was also performed (anti-
Hu IL-13 kit, Biosource, Camarillo, CA). Plates were coated with a polyclonal anti- human IL-13 (1.3 μg/ml), samples were added and then a biotin-labeled monoclonal antibody (MAb) to human IL-13 (1.5 μg/ml) (Biosource) was added followed by the addition of streptavidin-HRP (0.1 μg/ml) (Biosource) and TMB (KPL). Standard curves were generated using recombinant porcine IL-6 (Biosource, Camarillo, CA), IL-12 (R &
D Systems), IFN-γ, IL-4, IL-10 and IL-13 (Biosource). A computer-generated four- parameter curve-fit was used to calculate the concentration of each cytokine. The detection sensitivity limits for the reactions were as follows: 7 pg/ml for IL-6, IL-12,
IFN-γ, IL-4, IL-10, and IL-13.
242
4.3.15 Statistical analysis
The cytokine concentrations, ASC (PID 28 and 35) and CSC (PID 28 and 35)
numbers were compared among and within groups using Kruskal-Wallis rank sum test
(nonparametric). The seroconversion and coproconversion rates and the percentage of
pigs with diarrhea and shedding were compared using the Fisher’s exact test. Statistical
significance was assessed at P<0.05.
4.4 RESULTS
4.4.1 A/H phenotype of Gn pigs
All pigs were typed for the presence of the A and/or H antigen on their buccal
cells by hemagglutination inhibition (HI). Of all pigs used in the study, 16/30 (53 %) had
the A+/H+ phenotype and 13/30 (43%) had A+/H undetermined (?) phenotype. One of the
30 pigs (4%) had A-/H? type and belonged to the HuNoV-HS66VLP+ISCOM vaccinated
group of pigs that were euthanized at PID 28. All pigs, including controls challenged with
HuNoV-HS66 and euthanized at PID35/PCD7 had the A+/H+ phenotype.
4.4.2 The HuNoV-HS66 VLP vaccines protected pigs after HuNoV-HS66
challenge, with the VLP+mLT vaccine inducing complete protection
A subset of pigs in each group was euthanized at PID 28/PCD 0 and the other at
PID 35/PCD7, after receiving the HuNoV-HS66 challenge orally. Diarrhea and viral shedding were evaluated (Table 4.1). In the VLP+ISCOM vaccinated group, only 1/4
243 (25%) pigs had diarrhea for 1 day and in the VLP+mLT group none of the 4 pigs
developed diarrhea after HuNoV-HS66 challenge. However, 7/7 (100%) of the pigs in the
control group (ISCOM and mLT) had diarrhea after challenge. None of the pigs in either
vaccinated group had detectable virus shedding by RT-PCR, microwell hybridization or
antigen-ELISA; however, in the control group only 4/7 (57%) pigs shed virus. The mean
days of diarrhea and the mean diarrhea score for the VLP+ISCOM and the VLP+mLT
vaccinated groups were both significantly reduced when compared to the control group.
The Gn pigs in the VLP+ISCOM vaccinated group had a 75% protection rate against
diarrhea and those in the VLP+mLT group had 100%. Both groups had a 100%
protection rate against viral shedding.
4.4.3 The HuNoV-HS66 VLPs induced systemic and intestinal antibody
responses in Gn pigs regardless of the adjuvant used but the VLP+ISCOM
vaccine induced higher IgG antibody titers in serum pre-challenge and
higher IgM, IgA and IgG antibody titers pre- and also IgA post-challenge in
the IC of Gn pigs compared to the VLP+ISCOM vaccine
We assessed the immunogenicity of the HuNoV-HS66 VLPs, after oral/IN
inoculation of Gn pigs using ISCOM or mLT as adjuvants, and evaluated their protective efficacy against HuNoV-HS66 challenge. The Gn pigs receiving ISCOM matrix (n=6) or mLT (n=8), in the same number of doses and routes as the vaccinated groups, were combined as a control group (n=14).
244 The detailed antibody responses in the serum and IC are depicted in Figure 4.1
and 4.2 and the seroconversion and coproconversion rates are shown in Table 4.1. One- hundred-percent of the pigs that received either of the vaccine regimens (VLP+ISCOM or
VLP+mLT) had seroconverted to HuNoV-HS66 at virus challenge at PID 28/PCD 0
(Table 4.1). The IgM antibodies were first detected at PID 10 at low titers (GMT=12 and
9, respectively) after the oral priming dose VLP+ISCOM and VLP+mLT vaccines, and
increased significantly at PID 21 and 28 in both vaccinated groups, when compared to
controls. However at PID 35/PCD 7, the IgM antibody titers only in the VLP+mLT group
were significantly higher than controls.
The IgA antibodies were first detected in the serum at PID 21 in both
VLP+ISCOM and VLP+mLT groups (GMT=8 and 32, respectively), and were
significantly elevated at PID 28 and PID 35/PCD 7 in both vaccinated groups, when
compared to controls.
Serum IgG antibodies were first detected at low levels at PID 21, in both
vaccinated groups and were significantly higher than controls. Pre-challenge at PID
28/PCD0, the highest titers of IgG antibodies were detected in the VLP+ISCOM group
(GMT=381, range 20-2,560), compared to the VLP+mLT group (GMT= 44, range 5-
320). However post-challenge at PID 35/PCD 7, the IgG GMT remained the same in the
VLP+ISCOM group, although in one pig, the titer increased (640 to 2,560). In the
VLP+mLT group, the IgG antibody GMT increased 2-fold at PID 35/PCD 0 (GMT =
95), compared to that at PID 28 (GMT=44), and the post-challenge antibody titers were
significantly higher in both groups when compared to controls (Figure 4.1).
245 The IgM, IgA and IgG antibody titers were also measured in the IC of the pigs at challenge (PID 28/PCD 0) and post-challenge (PID 35/PCD 7) (Figure 4.2). One- hundred-percent of the pigs that received the VLP+ISCOM and 75% of those that received the VLP+mLT vaccine regimens coproconverted (PID 28/PCD 0) (Table 4.1) with either or both IgA and IgG antibodies in the IC. The VLP+ISCOM vaccine induced significantly higher levels of IgM, IgA and IgG antibody titers pre- and of IgA antibody titers post-challenge compared to the VLP+mLT vaccine (Figure 4.2). Serum antibody titers did not increase post-challenge (VLP+ISCOM), except for 1 pig that had diarrhea at
PCD 2 (GMT=40 to 160; 640 to 2,560, for IgA and IgG, respectively). Titers increased for both IgA and IgG (GMT=28 to 40; 5 to 34, respectively) in the VLP+mLT group at
PID 35/PCD 7 compared to those at PID 28/PCD 0, and both IgA and IgG antibodies were significantly increased post-challenge compared to controls.
4.4.4 The HuNoV-HS66 VLPs elicited higher numbers of ASC responses
locally (intestine) compared to systemically (spleen and blood) in the
VLP+ISCOM vaccinated pigs
The results of the ELISPOT assay for detection of virus-specific ASC in HuNoV-
HS66-inoculated and control Gn pigs euthanized at PID 28/PCD 0 and PID 35/PCD 7 are shown in Figure 4.3. The numbers of IgM, IgA and IgG ASC elicited in VLP+ISCOM and VLP+ mLT groups were significantly higher than in the controls at both PID 28/PCD
0 and PID35/PCD 7 for all tissues, except blood for the VLP+ISCOM group. At both
PIDs, higher numbers of IgA and IgG ASC were elicited locally (intestine) compared to systemically (spleen).
246 The highest numbers of IgA and IgG ASC were detected in the intestine of the
VLP+ISCOM group at PID 28 and 35, respectively (150 and 136; 150 and 157 ASC / 5 x
105 MNC, respectively).
At PID 28/PCD 0, significantly higher numbers of IgM ASC were detected in the
intestine of the VLP+ISCOM vaccinated pigs compared to the other groups and at PID
35/PCD 7 higher numbers (2-fold) were detected after HuNoV-HS66 challenge (from 59
to 136 ASC/ 5 x105 MNC). In the VLP+mLT vaccinated group, fewer IgM ASC (6 and
38, respectively) were detected at both PIDs. In spleen, pre- and post-challenge, the IgM
ASC numbers were also significantly higher in both vaccinated groups, compared to controls, although at lower numbers compared to those observed in intestine.
The IgA and IgG ASC numbers were also significantly higher in the intestine of the VLP+ISCOM pigs compared to the other groups, both at PID 28/PCD 0 and PID
35/PCD 7, although no boosting in the magnitude of response to either vaccine was observed post-challenge consistent with the protection observed. In spleen, the IgA and
IgG ASC numbers were significantly higher in both vaccine groups, compared to controls at both pre (PID 28/PCD 0) and post (PID 35/PCD 7) challenge. Significantly higher
numbers of IgA and IgG ASC were observed in blood (PBMC) of the VLP+mLT
vaccinated pigs pre-challenge compared to controls, and in both vaccinated groups post-
challenge.
247 4.4.5 Both VLP vaccines induced a significant increase in serum Th1 (IFN-γ)
concentrations only after viral challenge
The cytokine ELISA results are summarized in Figure 4.4. The pro-inflammatory cytokine (IL-6) was elevated (but not significantly) in the serum of both vaccinated groups at PID 28/PCD 0 and at PID 35/PCD 7. At PID 30/PCD 2, a non-significant transient increase in IL-6 was detected in the control group. The Th1 cytokine (IL-12) was detected constitutively in the serum of pigs from all groups at birth. It was significantly elevated in the serum of only the VLP+mLT group at PID 28/PCD 0, compared to the other groups. A transient (but not significantly elevated) peak of the Th1 cytokine IFN-γ was detected in serum of the VLP+mLT group at PID 2 (after one oral dose of the vaccine), with a significant increase only post-challenge at PID 35/PCD 7.
For the ISCOM+VLP vaccinated pigs, a low peak of IFN-γ was detected at PID 28/PCD
0 and increased significantly after challenge at PID 30/PCD 2 and at PID 35/PCD 7, compared to controls.
The Th2 cytokine IL-4 was significantly elevated in the serum of both vaccinated groups at PID 2 and 7 compared to controls and in the serum of the VLP+mLT pigs at
PID 10 and 21, compared to the other groups. The other Th2 cytokine IL-13 was significantly elevated in the serum of only the VLP+mLT vaccinated pigs at PID 28/PCD
0 (64 pg/ml) and in the serum of both vaccinated groups post-challenge (PID 30/PCD 2 and 35/PCD 7), although not significantly, compared to controls.
248 The Th2-T-reg cytokine (IL-10) peaked (52 pg/ml) at PID 10 in the serum of the
VLP+ISCOM pigs and later (57 pg/ml) at PID 28/PCD 0 in the VLP+mLT group.
Higher, but not significantly, levels of IL-4 and IL-10 were also detected in serum of the
control group after viral challenge at PID 35/PCD 7.
4.4.6 The Th1 cytokine (IFN-γ) was significantly elevated post-challenge in
IC only in the VLP+mLT vaccinated pigs
The cytokine concentrations were measured in the IC of the pigs after euthanasia
(PID 28/PCD 0 and PID 35/PCD7) and are depicted as mean concentrations of each
cytokine in the IC of each group (Figure 4.5). Low (IL-6, IL-12, IL-13) to moderate (IL-4
and IL-10) cytokine concentrations were detected in the IC of both vaccinated groups at
both PID/PCDs, although not significantly higher than those in the IC of the controls.
However, significantly higher concentrations (166 pg/ml) of the Th1 cytokine (IFN-γ)
were detected in the IC of the VLP+mLT group one week post-challenge (PID 35/PCD
7), when compared to the other groups.
249 4.4.7 Th1 (IL-12 and IFN-γ), Th2 (IL-4), Th2/T-reg (IL-10) and pro-
inflammatory (IL-6) CSC were elicited at higher numbers systemically only
in the VLP+mLT vaccinated Gn pigs pre-challenge
Generally, higher numbers of CSC were elicited in both vaccinated groups
compared to controls post-challenge (Fig 4.6 and 4.7). However, only in systemic tissues
(spleen and PBMC) and only in the VLP+mLT pigs were IL-6 and IFN-γ significantly elevated (28 and 23 CSC/ 5 x 105 MNC; 58 and 25 CSC/ 5 x 105 MNC, respectively).
pre-challenge. The IL-12 was also significantly elevated in spleen and IL-10 in PBMC of
the VLP+mLT pigs pre-challenge.
Post-challenge, the highest numbers of the pro-inflammatory IL-6 CSC were
detected in blood (PBMC) of both vaccinated groups, although not significantly higher
compared to controls. Significantly higher numbers of Th1 (IL-12 and IFN-γ) CSC were
observed in the VLP+ISCOM (140 and 98 CSC/ 5 x 105 MNC, respectively) and in the
VLP+mLT (328 and 39 CSC/ 5 x 105 MNC, respectively) vaccinated pigs, compared to
controls. Significantly higher numbers of Th2 (IL-4) CSC were detected only in the
VLP+ISCOM pigs in all tissues (34-48 CSC/ 5 x 105 MNC), compared to the other
groups and only IL-4 CSC were significantly elevated in the intestine. Significantly
higher numbers of Th2/T-reg (IL-10) CSC were observed in blood (PBMC) of both
vaccinated groups (320 CSC/ 5 x 105 MNC in mLT+VLP and 663 CSC/5 x 105 MNC in
ISCOM+VLP), compared to controls.
250 4.5 DISCUSSION
The HuNoV infections are usually self-limiting and transient; however, the
increasing numbers of NoV outbreaks worldwide and their impacts in settings such as nursing homes, hospitals, and the military indicate that the development of effective vaccines would be beneficiary (18, 28). The HuNoV-HS66 strain replicates and causes diarrhea in Gn pigs (9), and we have recently evaluated the immune responses to a GII.4
HuNoV (HS66 strain) in the Gn pig model (M. Souza, S. M. Cheetham, M. S. P.
Azevedo, V. Costantini, and L. J. Saif, submitted for publication). In this study, we
evaluated the immunogenicity of HuNoV-HS66 VLPs administered in three doses (one
oral and 2 IN) in conjunction with each of two adjuvants (ISCOM or mLT), and
evaluated protection elicited by both vaccine regimens against HuNoV-HS66 oral
challenge.
None of the vaccinated pigs in either the VLP+ISCOM or VLP+mLT groups shed
virus and the protection rate against diarrhea was 75% and 100%, respectively. Post-
challenge, only 57% of control pigs shed detectable virus and 57% and 43%
coproconverted with IgM and IgA antibodies at PID 35/PCD 7, respectively. A previous
study in our lab (8) showed that the pigs with the A+ and/or H+ phenotype had increased
diarrhea and viral shedding rates compared to non- A+ and H+ Gn pigs after infection
with the GII.4 HuNoV-HS66 strain. Because all the pigs in the study were typed for the
A/H phenotype and all pigs that were challenged with HuNoV had A+/H+ phenotype, we
cannot conclude that the A/H phenotype influenced the outcome of the study. However,
the older age at which the Gn pigs were challenged could have played a role in the lack of virus shedding detection in the control group. In our previous studies we inoculated the
251 pigs at an earlier age (3-to-7-day-old compared to 35-day-old) and maybe they are more
resistant to infection at an older age. However, because almost all pigs in both ISCOM
and mLT control groups had diarrhea compared to only 1 pig in the VLP+ISCOM group,
we assume that viral replication, undetected at very low levels, may have occurred in the
control pigs.
The immunogenicity of HuNoV VLPs, administered alone or with mLT, has been
evaluated in mice, in which the VLPs induced both systemic and mucosal (fecal and
vaginal) immune responses (13). The immune responses to NV VLPs have also been
determined in human volunteers. However, previous exposures to HuNoV can be confounding variables in interpretating the magnitude of the immune responses elicited
by these VLPs. Therefore, Gn animals, such as pigs and calves constitute valuable
models for the study of both local and systemic immune responses to animal NoV VLPs
(14) and HuNoV VLPs.
In our study, both vaccine regimens (VLP+ISCOM and VLP+mLT) induced
100% seroconversion to HuNoV-HS66 VLPs at PID 28, with the highest titers of IgA and IgG serum antibodies detected in the VLP+ISCOM group. However, the IgA and
IgG antibody responses were boosted in the VLP+mLT group after viral challenge (PID
35/PCD 7), although the GMT remained lower than those of the VLP+ISCOM group.
The high seroconversion rates observed in our study are very similar to those previously
observed in humans (90%) (30) and in mice (7) orally immunized with 2 or 3 doses of
NV VLPs. Serum antibody responses were also induced in Gn calves immunized with
bovine norovirus (BoNoV) VLPs and various adjuvants, including mLT and ISCOM, and routes of inoculation (14). Low to moderate serum IgG antibody titers (GMT= 60 to 100)
252 were detected pre-challenge in calves immunized IN with BoNoV VLP+mLT. Post-
challenge, the highest increases in serum IgG antibody titers were also detected in this
group, followed by the VLP+mLT/oral vaccinated calves (14).
In intestinal contents, we detected IgM, IgA and IgG antibodies in both
vaccinated groups pre- (PID 28/PCD 0) and post-challenge (PID 35/PCD7). The IgM,
IgA and IgG antibody titers were significantly elevated in the VLP+ISCOM group pre-
challenge, compared to the other groups. Mucosal (from saliva, feces, vaginal and semen washes) anti-NV VLP IgA was also observed in about 35% of the individuals orally immunized with the same dose of NV VLP (without adjuvant) as used in this study (30).
However in our study, the coproconversion rate (IgA or IgG antibodies in IC) pre- challenge was higher (75% in the VLP+mLT group and 100% in the VLP+ISCOM group) than for the non-adjuvanted NV VLPs in humans, likely reflecting the enhanced intestinal responses by mucosal adjuvants. The highest antibody titers in the IC were IgA in the VLP+ISCOM group pre- and post-challenge. These results differed from results of the BoNoV VLP study where fecal IgA antibodies were detected only in 2/4 calves that received either 2 or 3 doses of VLP+mLT/IN. In contrast to our pig studies, no fecal IgG was detected pre-challenge in any of the calves in the VLP+ISCOM or VLP+mLT (oral or IN) inoculated groups. However, fecal IgG antibodies were elicited post-challenge with BoNoV in calves from all vaccination regimens (14). In the present study, although the highest titers of IgA and IgG antibodies were detected pre-challenge in IC of the
VLP+ISCOM group, increases in IgA and IgG antibodies from PID 28/PCD 0 to PID
35/PCD 7 were observed in the VLP+mLT group.
253 To assess intestinal and systemic ASC responses, IgM, IgA and IgG ASC were
evaluated in the intestine, spleen and blood (PBMC) of subsets of pigs from all groups
both pre- and post-challenge. The highest numbers of ASC of all three isotypes were
detected locally (intestine) in the VLP+ISCOM vaccinated pigs, reinforcing that the
administration of VLP by the oral plus IN combined routes constitutes an efficient
vaccine strategy likely mimicking the natural routes of HuNoV infection and also
reflecting the particulate and immunogenic nature of the VLPs. The magnitude of the
ASC responses in the intestine however, was not boosted by the HuNoV-HS66 challenge,
likely reflecting the high degree of protection observed (no virus shedding). Because 10- fold higher pre-challenge ASC numbers were elicited in the intestine of vaccinated pigs in this study, compared to those detected at PID 28 in naïve Gn pigs orally inoculated with the same HuNoV-HS66 challenge inoculum (M. Souza, S. M. Cheetham, M. S. P.
Azevedo, V. Costantini, and L. J. Saif, submitted for publication), more than one dose of
HuNoV non-replicating VLP vaccines may be needed to elicit strong immune responses and possibly protection (30).
Low to moderate, but significantly elevated, IgM, IgA and IgG ASC responses were detected post-challenge systemically (spleen and blood) in both vaccinated groups compared to controls, and were induced at a higher magnitude in the VLP+mLT group compared to the VLP+ISCOM group (except for IgM and IgG in PBMC). Systemic IgA
ASC responses were also detected from the PBMCs of volunteers who were orally vaccinated with NV VLPs and the numbers of ASC elicited did not differ significantly among different VLP doses (250 μg to 2000 μg). However, unlike in the pig model, the intestinal ASC responses could not be evaluated in the human volunteers (30).
254 The Th1 (IL-12 and IFN-γ), Th2 (IL-4 and IL13) and Th2/T-reg (IL-10) responses were induced in the serum of both vaccinated groups, mainly after the second dose of vaccine; however, the concentrations were significantly higher for Th1 (IFN-γ) cytokine in both vaccinated groups post-challenge. In a human volunteer study, the IFN-γ levels were measured in the PBMC culture supernatants of NV-VLP vaccinees after in vitro NV-VLP stimulation, and significant increases in IFN-γ production were detected in
PBMC collected 21 days after the first oral NV VLP immunization, in comparison to pre- immunization levels; however, no IFN-γ responses were detected in the PBMC supernatant of volunteers that received a high dose of VLP (2000 µg), and the authors suggested that the lack of response could be due to tolerance induced by the high VLP dose (30).
The Th2 (IL-4) concentrations were significantly higher in the serum of both vaccinated groups at PID 2, 7 and 21, and in the serum of the VLP+mLT group at PID
10, compared to the other groups. In the adult volunteer study in which IFN-γ was detected in the PBMC culture supernatant, the IL-4 concentrations were also evaluated.
Because no IL-4 was detected, the authors concluded that a dominant Th1 response was induced by the NV VLPs (30). However, this must be interpreted carefully because other
Th2 cytokines exist that were not evaluated; therefore, a more complete panel of cytokines should be evaluated before this conclusion is reached. We also evaluated the concentrations of another Th2 cytokine (IL-13) in the serum of the Gn pigs and significantly higher levels were detected at PID 28 and PID 30/PCD2 in the VLP+mLT group, when compared to the other groups. Higher levels, although not significantly were also observed in serum of both vaccinated groups post-challenge. The IL-13 plays an
255 important role in the modulation of the immune responses, especially to intracellular
parasites (34). Interestingly, the increased levels of IL-13 post-challenge (PID 35/ PCD 7)
in the serum of both vaccinated groups paralleled that of the pro-inflammatory IL-6
cytokine, perhaps reflecting the effect of IL-6 in inducing the anti-inflammatory IL-13.
Peaks of the Th2/T-reg (IL-10) cytokine, although not statistically significant compared
to controls, were detected pre-challenge in serum of the VLP+ISCOM pigs at PID 10 and
later in serum of the VLP+mLT (PID 28/PCD 0) but in the control groups only post-
challenge (PID35/PCD 7).
In IC, the only cytokine that was significantly elevated was Th1 (IFN-γ) in the
VLP+mLT pigs post-challenge. The high concentration (166 pg/ml) of IFN-γ in the IC of the VLP+mLT pigs corroborates those concentrations found in serum and likely represents the boosted memory Th1 responses previously elicited by the HuNoV VLPs
(30). The IFN-γ was also detected in the VLP+ISCOM group although at much lower and non-significantly elevated levels.
The pro-inflammatory (IL-6), Th1 (IL-12 and IFN-γ), Th2 (IL-4 and IL-13) and
Th2-T-reg (IL-10) CSC responses were also evaluated both locally (intestine) and systemically (spleen and blood). To our knowledge, intestinal CSC responses have not been examined previously for NoV infection in any species. The VLP+mLT group had higher pro-inflammatory, Th1 and Th2/T-reg CSC numbers in systemic tissues pre- challenge, compared to the VLP+ISCOM group. This CSC profile pre-challenge coincided with complete protection against virus shedding and diarrhea observed in this group.
256 Post-challenge, significantly higher numbers of IL-12 and IFN-γ CSC were detected in spleen of both vaccinated groups, when compared to controls. Although the increase in IFN-γ numbers from pre- to post-challenge in the VLP+ISCOM group, both systemically and intestinally, suggests a boosting of the memory response to HuNoV-
HS66 VLP after viral challenge, the increases were not significant, except for the CSC numbers in the spleen at PID 35/PCD 7, consistent with the high degree of protection induced upon HuNoV challenge.
The Th2 (IL-4) CSC numbers were significantly elevated post-challenge only in the VLP+ISCOM group, both locally (intestine) and systemically, when compared to the other groups. In comparison, no IL-4 secretion by CSC isolated from the blood of human volunteers previously immunized with one or two doses of NV VLPs was detected (30).
Because the IL-4 CSC responses were enhanced only in the VLP+ISCOM group, this suggests that ISCOM may influence this Th2 response compared to the lack of adjuvant with NV VLPs in human trials.
In conclusion, both VLP+ISCOM and VLP+mLT vaccine regimens induced high rates of seroconversion, coproconversion and protection against viral shedding and diarrhea when compared to the controls. Although not all control pigs shed virus, the majority of them had diarrhea with significantly higher diarrhea scores compared to the vaccinated pigs. The Th1 (IL-12 and IFN-γ) cytokines were elicited at higher levels in the serum of both vaccinated groups with low to moderate levels of the pro-inflammatory
(IL-6) and Th2/T-reg (IL-4, IL-10 and IL-13) also elicited in serum by both vaccination regimens. The significantly increased IFN-γ concentrations in the IC of the VLP+mLT group after challenge suggests a booster Th1 response to HuNoV induced by viral
257 challenge. Higher systemic CSC responses were mainly observed in the VLP+mLT vaccinated group, and these responses coincided with the higher protection rate against virus shedding observed in this group. Pre-challenge, the VLP+ISCOM vaccine induced higher IgA and IgG ASC both systemically and locally and IgA and IgG in IC with significantly elevated Th2 (IL-4) intestinal CSC post-challenge. In contrast, the
VLP+mLT induced higher systemic Th1 and Th2/T-reg CSC numbers pre-challenge and the highest Th1 cytokine responses in IC. Collectively, these results indicate that
VLP+mLT induced a more balanced Th1/Th2 response pre-challenge, whereas the
VLP+ISCOM induced a more Th2 biased response pre-challenge but with significantly higher systemic Th1 (IFN-γ) responses post-challenge. However, both vaccines induced protection upon challenge with HuNoV, although protection against diarrhea was less efficient in the latter group.
To our knowledge this is the first study to evaluate the immunogenicity and protective potential of GII HuNoV VLPs and also to assess, in detail, both local and systemic immune responses to HuNoV VLP vaccines prior and subsequent to homologous viral challenge in an experimental animal model.
4.6 ACKNOWLEDGMENTS
We acknowledge Sonia Cheetham for producing the recombinant baculovirus containing the HuNoV-HS66 capsid and the guinea pig anti-HS66 hyperimmune serum.
We also thank Dr. J Hanson, Mr. R. McCormick and Ms. J. McCormick for animal care;
Dr. J. H. Hughes, who kindly provided the original NoV GII.4 human fecal sample, Karin
Lovgren-Bengtsoon and Bror Morein (Swedish, University of Agricultural Sciences,
258 Uppsala, Sweden) who kindly provided the ISCOM, and J. Clements (Tulane University
Medical Center, New Orleans, La.) who kindly provided the R192G mLT.
Salaries and research support were provided by state and federal funds
appropriated to the Ohio State University. This work was supported by a grant from the
National Institute of Allergy and Infectious Diseases, National Institutes of Health, grant
No. R01-AI49742 to the corresponding author (L. J. Saif). Menira Souza was a fellow of
Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Brasilia,
Brazil, from July-2002 to July 2005.
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264
Seroconversiond Protection Protection Vaccine Virus Diarrhea Diarrhea (%)/GMT range against against Coproconversionf # pigs shedding # pigsb (mean days) (mean score)c at PID 28 and 35 diarrheae sheddinge (%) at PID 28 # pigs a (%) (%)
A VLP+ISCOM 100 0 1 0.3Bg 3B 75 100 100A (n= 4) (20-2,560; 20-2,560)
VLP + mLT 100 A 0 0 0B 2B 100 100 75A (n= 4) (40-640; 20-2,560) Controls 4 7 2A 10A 0B 0 NAe 43B
265 (n=7) aVirus shedding was detected by RT-PCR and hybridization assay after HuNoV-HS66 challenge (PID28-35) bDiarrhea present if fecal swab scores were ≥2 after HuNoV-HS66 challenge cRepresents the sum of daily rectal swab scores (PCD 1-6) of each pig divided by the number of pigs in that group dSeroconversion and coproconversion were determined by immunocytochemistry at PID 28, values in parentheses reflect the GMT range among pigs. eProtection rate was calculated based on the reduction of virus shedding or diarrhea, compared to controls. Fifty-seven percent of the pigs in the control groups shed virus after HuNoV-HS66 challenge fCoproconversion with either IgA or IgG antibodies at PID 28. gValues in columns with different superscript letters (A or B) differ significantly (p<0.05)
Table 4.1. Fecal virus shedding, diarrhea, seroconversion, protection rates and coproconversion in Gn pigs vaccinated with either HuNoV-HS66 VLP+ISCOM, HuNoV-HS66 VLP+mLT or controls (ISCOM and mLT) Serum IgM Ab A 1000 Serum IgG Ab 250 250 Serum IgA Ab A 200 200 750 150 150 500 GMT GMT
GMT 100 A 100 A 250 A A 50 A A 50 B 0 B 0 0 B B B B BBC 0 10 21 28/0 35/7 0 10 21 28/0 35/7 0 10 21 28/0 35/7
PID/PCD PID/PCD PID/PCD
Figure 4.1. Isotype-specific (IgM, IgA and IgG) antibody responses in the serum of gnotobiotic pigs vaccinated with 1 oral and 2 IN doses of each vaccination regimen
(HuNoV-HS66VLP+ISCOM or mLT) or controls. Symbols: HuNoV-HS66VLP+ISCOM, V HuNoV-HS66VLP+mLT controls (ISCOM or mLT)
Different letters denote statistical significance (P<0.05). 266
150 IgM Ab in IC 300 IgA Ab in IC A 150 IgG Ab in IC 125 A 250 A 125 A B A 100 200 100 75 75 150 A GMT GMT GMT 50 50 100 B 25 50 25 B B B B C B B 0 0 0 28/0 35/7 28/0 35/7 28/0 35/7 PID/PCD PID/PCD PID/PCD
Figure 4.2. Isotype-specific (IgM, IgA and IgG) antibody responses in the IC of gnotobiotic pigs vaccinated with 1 oral and 2 IN doses of each vaccination regimen
(HuNoV-HS66VLP+ISCOM or mLT) or controls. Symbols: HuNoV-HS66+ISCOM; HuNoVVLP+mLT; controls.
Different letters denote statistical significance (P<0.05) IgA ASC in intestine 250 IgM ASC in intestine 250 IgG ASC in intestine A 250 200 A A A A 200 200 150 150 150 100 A 100 B B 100 B 50 B C 50 B B C C C 50 0 0 0 C C ASC/ 5 x 105 MNC ASC/ ASC/ 5 x 105 MNC ASC/
28/0 35/7 5 x 105 MNC ASC/ 28/0 35/7 28/0 35/7 PID/PCD PID/PCD PID/PCD
IgM ASC in spleen IgA ASC in spleen 50 IgG ASC in spleen 50 50 A A A B 25 B 25 A 25 A A B B B B C C C C 267 C 0 C 0 5 x 105 MNC ASC/ 0 ASC/ 5 x 105 MNC ASC/ 28/0 35/7 28/0 35/7 5 x 105 MNC ASC/ 28/0 35/7 PID/PCD PID/PCD PID/PCD
IgG ASC in PBMC IgM ASC in PBMC IgA ASC in PBMC A 50 100 50 75 A A A 25 A A 50 25 A A 25 A B B B B 5 x 105 MNC ASC/ B B B
B 5 x 105 MNC ASC/ B 0 0 0 ASC/ 5 x 105 MNC ASC/ 28/0 35/7 28/0 35/7 28/0 35/7 PID/PCD PID/PCD PID/PCD
Figure 4.3. Mean numbers of isotype-specific (IgM, IgA and IgG) antibody secreting cells in the intestine, spleen and PBMC of Gn pigs
vaccinated with 1 oral and 2 IN doses of HuNoV-HS66VLP+ISCOM or HuNoV-HS66VLP+mLT. Symbols: HuNoV-HS66VLP+ISCOM
HuNoV-HS66VLP+mLT; controls. Different letters denote statistical difference (P<0.05) IL-12 in serum Th1 250 250 IFN-γ in serum 200 200 A A A 150 150 B pg/ml 100 100 pg/ml 50 50 B B 0 0 0 2 7 10 21 28/0 30/2 35/7 0 2 7 10 21 28/0 30/2 35/7 PID/PCD PID/PCD Pro-inflammatory 100 IL-6 in serum 75 50
pg/ml 25 268 0 0 2 7 10 21 28/0 30/2 35/7 PID/PCD Th2/T-reg IL-4 in serum IL-13 in serum 150 IL-10 in serum 150 150 100 100 A 100 A 50 A 50 50
A A pg/ml pg/ml B A pg/ml 0 B 0 0 B B B B 0 2 7 10 21 28/0 30/2 35/7 0 2 7 10 21 28/0 30/2 35/7 0 2 7 10 21 28/0 30/2 35/7 PID/PCD PID/PCD PID/PCD
Figure 4.4. Cytokine concentrations in the serum of Gn pigs vaccinated with 1 oral and 2 IN doses of each vaccination regiment (HuNoV-HS66VLP+ISCOM or mLT) or controls.
Symbols: HuNoV-HS66VLP+ISCOM;V HuNoV-HS66VLP+mLT; controls. Different letters denote statistical significance (P<0.05). Th1 IL-12 in IC 50 IFN-γ in IC 200 * 150 25 100 pg/ml
pg/ml 50 0 0 28/0 35/7 28/0 35/7 PID Pro-inflammatory PID IL-6 in IC 50
25 269 pg/ml 0 28/0 35/7 PID Th2/T-reg IL-4 in IC IL-13 in IC IL-10 in IC
200 50 100 150 40 30 100 20 50 pg/ml pg/ml 50 10 pg/ml 0 0 0 28/0 35/7 28/0 35/7 28/0 35/7 PID PID PID
Figure 4.5. Th1 (IFN-γ and IL-12), pro-inflammatory (IL-6), Th2 (IL-4) and Th2/T-reg (IL-10) cytokine concentrations in the intestinal contents of Gn pigs
vaccinated with 1 oral and 2 IN doses of each vaccination regimen (HuNoV-HS66-VLP+ISCOM or mLT) or controls. Symbols:
HuNoV-HS66VLP+ISCOM; HuNoV-HS66VLP+mLT; controls *significantly higher than the other groups (P<0.05). Th1
IL-12 CSC in intestine IL-12 CSC in spleen IL-12 CSC in PBMC 400 400 400 A MNC MNC MNC
5 300 5 300 5 300 B 200 200 200 A C 100 100 100 B B 0 0 0 CSC/ 5 x 10 CSC/ 5 x 10 CSC/ 5 x 10 28/0 35/7 28/0 35/7 28/0 35/7 PID/PCD PID/PCD PID/PCD IFN-γ CSC in PBMC IFN-γ CSC in intestine IFN-γ CSC in spleen 200 200 200 A MNC MNC 150 150 MNC 5 150 5 5 100 100 A 100 B A 50 50 50 B B C B B 0 0
270 0 CSC/ 5 x 10 CSC/ 5 x 10 CSC/ 5 x 10 28/0PID/PCD 35/7 28/0PID/PCD 35/7 28/0PID/PCD 35/7 Pro-inflammatory
IL6 CSC in intestine IL6 CSC in PBMC 100 100 IL6 CSC in spleen 100 MNC MNC 75 75 MNC 75 5 5 5 50 50 A 50 A 25 25 25 A B B B 0 0 0 CSC/ 5 x 10 CSC/ 5 x 10 28/0 PID/PCD 35/7 28/0PID/PCD 35/7 CSC/ 5 x 10 28/0PID/PCD 35/7
Figure 4.6. Mean numbers of Th1 (IFN-g and IL-12) and pro-inflammatory (IL-6) cytokine secreting cells in intestine, spleen and PBMC of Gn pigs vaccinated
with each vaccination regimen (HuNoV-HS66VLP+ISCOM or mLT) or controls. Symbols: HuNoV-HS66VLP+ISCOM; HuNoV-HS66VLP+mLT; controls
Different letters denote statistical significance (P<0.05) Th2/T-reg
IL-4 CSC in spleen 200 IL-4 CSC in intestine IL-4 CSC in PBMC 200 200
MNC 150 MNC 150
5 150 5 MNC 100 100 5 100 A A A 50 B B 50 B B 50 B B 0 0 0 CSC/ 5 x 10 28/0 35/7 CSC/ 5 x 10 28/0 35/7 28/0 35/7 PID/PCD PID/PCD CSC/ 5 x 10 PID/PCD 271
IL-10 CSC in intestine IL-10 CSC in spleen IL-10 CSC in PBMC 1000 1000
MNC 750 1000 5
MNC 750 A MNC 5 750
500 5 500 B 500 A C 250 250 250 B 0 0 B CSC/ 5 x 10 0
28/0 35/7 CSC/ 5 x 10 28/0 35/7 CSC/ 5 x 10 28/0 35/7 PID/PCD PID/PCD PID/PCD
Figure 4.7. Mean numbers of Th2 (IL-4) and Th2/T-reg (IL-10) cytokine secreting cells in intestine, spleen and PBMC of Gn pigs vaccinated
with each vaccination regimen (HuNoV-HS66VLP+ISCOM or mLT) or controls. Symbols: HuNoV-HS66VLP+ISCOM; HuNoV-HS66VLP+mLT; controls
Different letters denote statistical significance (P<0.05) CHAPTER 5
GENERAL SUMMARY AND CONCLUSIONS
Human noroviruses (HuNoV) are transmitted by the fecal-oral route and constitute the main non-bacterial pathogens involved in epidemic food- and water-borne outbreaks worldwide, causing a major disease burden in both developed and developing countries, and affecting people from all ages. They cause a self-limiting disease that lasts, in most cases, up to 48 hours and it is characterized by nausea, vomiting and abdominal cramps. However, long term viral shedding and profuse diarrhea are common complications in certain populations such as immmunocompromised patients and the elderly.
The HuNoV have extensive genetic and antigenic variability and this variability contributes to the susceptibility of the host to multiple infections throughout their lives. It also poses problems when developing an efficacious vaccine that will provide protection against multiple strains circulating worldwide, which would be particularly useful in highly susceptible populations such as the elderly and the military. They are generally highly stable in the environment, leading to a high rate of environmental contamination and person-to- person spread which contributes to their dissemination and consequently to disease impact.
Because these are fastidious viruses that do not grow in routine cell cultures elucidating their pathogenesis, replication, viral-host interactions, host immune responses and vaccine development has been hampered. Moreover, although some genetic factors such as
272 the histo-blood group antigens and secretor status of an individual were identified as genetic
factors associated with risk of GI.1 Norwalk virus and GII.4 HuNoV (HS66 strain) in humans
and in pigs, respectively, the determinants of susceptibility and/or resistance to HuNoV are
still unclear.
Because recent important progress has been made by the in vitro growth of GI and
GII HuNoV strains, using a 3-dimensional organoid model of human intestinal epithelium, and by the successful replication of the HuNoV GII.4-HS66 strain in the gnotobiotic (Gn) pig model, these in vitro and in vivo systems can now be used to study other aspects of HuNoV infections. More importantly, the knowledge derived from these latest advances can be used to perfect those systems and also to find more suitable alternatives.
In this study we evaluated two animal models (Gn pigs and calves) for the study of the local and systemic immune responses to a GII.4 HuNoV (HS66 strain) and also to assess the immune responses of Gn pigs to a vaccine composed of HuNoV-HS66 VLPs adjuvanted with either ISCOM or mLT, both prior to and after homologous virus challenge.
Our first objective was to delineate the local and systemic immune responses of Gn pigs after infection with the HuNoV-HS66 strain, compared to mock-inoculated controls. We observed that although, IgM, IgA and IgG antibodies were elicited at low titers 65% of the
HuNoV-HS66-infected pigs seroconverted at post-inoculation day (PID) 28 with either IgA or IgG antibodies. Sixty-four percent of the pigs had IgA or IgG antibodies in their intestinal contents (IC), also at low levels.
The local and systemic antibody-secreting cell (ASC) responses were also evaluated and low numbers of both IgA and IgG ASC were detected both locally and systemically with similar numbers of IgA and IgG ASC detected both locally and systemically at PID 21 or 28.
273 In serum, delayed innate (IFN-α) and low, early pro-inflammatory (IL-6) responses were detected, in concert with a balanced Th1/Th2 response. In IC only innate (IFN-α) (early and late) and Th1 (IL-12) (late) responses were significantly higher compared to controls.
The local and systemic cytokine-secreting cell (CSC) responses were characterized by low numbers of the pro-inflammatory (IL-6) CSC detected early in the gut and later systemically
(blood), and a generally more biased Th1 (IL-12 and IFN-γ) response both locally and systemically.
Our second objective was to evaluate the pathogenicity of the HuNoV-HS66
strain in Gn calves and to evaluate their local and systemic immune response to this GII.4
HuNoV strain. The HuNoV-HS66 replicated in the proximal portion of the small
intestine (jejunum) and caused diarrhea and more pronounced intestinal lesions during the acute phase of infection, compared to those observed in the Gn pigs. Viral shedding was detected from post-inoculation day 1-6 and 67% of the animals seroconverted with low levels of HuNoV-HS66-specific IgA and IgG antibodies. Moderate numbers of IgA and IgG ASC were also observed at PID 28 both locally and systemically, with higher numbers of IgG ASC detected in intestine, compared to those observed in the Gn pigs. A balanced Th1 (IL-12 and IFN-γ)/Th2/T-reg (IL-10) response was observed in serum, fecal samples and among MNC populations in the gut, spleen and blood. Pro- inflammatory cytokine (TNF-α) was induced in the serum and fecal samples of the
HuNoV-HS66-inoculated calves, and this cytokine was the only one that increased
significantly in the IC of the Gn calves, corroborating the higher intestinal pathology
induced in these animals.
274 Our third objective, was to evaluate the systemic and local immune responses of Gn
pigs to one oral followed by two IN booster doses of HuNoV GII.4 HS66 strain (HuNoV-
HS66) VLP vaccine adjuvanted with either mLT or ISCOM mucosal adjuvants, both prior to
and after viral challenge with homologous virus, and also the induction of protection in the
vaccinated pigs, compared to controls (only ISCOM or mLT).
A 100% seroconversion rate was observed in the vaccinated pigs, regardless of the
vaccine regimen, and coproconversion was detected in the VLP+ISCOM pigs compared to
75% of the VLP+mLT group. However, only 57% of the control pigs shed virus post-
challenge. The VLP+ISCOM vaccine induced higher IgA and IgG antibody titers both pre-
and post-challenge in serum and in IC, except for IgG antibodies in IC post-challenge. It also
induced higher IgA and IgG ASC locally both pre- and post-challenge compared to the
VLP+mLT vaccine, whereas the VLP+mLT elicited systemic (spleen) IgA and IgG ASC
both pre-and post-challenge (Table 5.1).
In serum, a peak of Th1 (IFN-γ) was induced by both vaccination regimens post-
challenge, showing that priming by both vaccines occurred prior to boosting of memory by
the homologous viral challenge; however a significant increase of IFN-γ in IC was only observed in the VLP+mLT vaccinated pigs. Higher numbers of systemic (spleen) IL-12 CSC both pre- and post-challenge were elicited by the VLP+mLT vaccine, and higher numbers of
IFN-γ pre-challenge and of Th2/T-reg (IL-10) pre-challenge (blood), showing a balanced
Th1/Th2 response. The VLP+ISCOM vaccine regimen induced a more Th2 biased response eliciting higher IgA antibody levels in both serum and IC pre- and post-challenge, higher numbers of IgA ASC in intestine and with higher numbers of IL-4 CSC post-challenge both locally (intestine) and systemically (spleen and blood) compared to the VLP+mLT regimen
(Table 5.2).
275 A summary of the results of this study are depicted in Tables 5.1 and 5.2. As
observed in Table 5.1, both vaccine regimens using multiple doses (1 oral priming and 2 IN
boosting doses) elicited higher IgA and IgG seroconversion and coproconversion rates at PID
28, compared to only one oral dose of HuNoV-HS66, and the VLP+ISCOM vaccine elicited
higher IgA and IgG antibody titers in serum and in IC both pre- and post- challenge
compared to the VLP+mLT regimen. The ASC numbers were also elicited at higher levels in
the vaccinated pigs at PID 28, compared to those elicited by only one oral dose of HS66, and
the VLP+ISCOM vaccine elicited higher numbers of IgA and IgG ASC in gut pre- and post-
challenge. In comparison, the VLP+mLT vaccine elicited higher IgA and IgG ASC numbers
in spleen post-challenge and higher IgA and IgG ASC numbers in blood pre-, and IgA, post-
challenge compared to the VLP+ISCOM vaccine.
The cytokine levels in serum IC and the CSC responses are shown in Table 5.2. The
HuNoV-HS66 single oral dose induced early low peaks of pro-inflammatory (IL-6), Th1
(IFN-γ), Th2 (IL-4), Th2/T-reg (IL-10). However, the magnitude of the pro-inflammatory
(IL-6), Th1 (IL-12), Th2 (IL-4) and Th2/T-reg (IL-10) elicited by one oral dose of HuNoV-
HS66 was similar to that induced by oral priming and 2 IN doses of VLP+ISCOM and
VLP+mLT. At PID 28, higher levels of Th1 (IFN-γ) were elicited by the VLP+ISCOM vaccine compared to either one oral dose of HS66 or by the VLP+mLT vaccine, although at
PID 35/PCD7 both vaccines elicited high levels of IFN-γ in the serum. These data show that
priming by the vaccines occurred and that the homologous viral challenge induced a boost in the immune responses in both groups.
Although the VLP+ISCOM regimen induced higher Th2 (IL-4) earlier (PID 2, 7) in serum, the VLP+mLT elicited later IL-4 peaks (PID 10 and 21) and also significantly higher
Th2 (IL-13) at PID 28, compared to the VLP+ISCOM. The Th1 (IL-12) cytokine was
276 elevated in the IC of the pigs that received a single oral dose of HuNoV-HS66 at PID 28. In comparison, the Th1 cytokine IFN-γ was detected at high concentrations (10-fold higher than those found in the IC of the HuNoV-HS66 at PID 28), post-challenge, only in the IC of the
VLP+mLT vaccinated pigs.
Generally the CSC were elicited at higher numbers at PID 28 in both vaccination groups compared to one oral dose of HuNoV-HS66, except for IL-12 in spleen and blood and
IFN-γ in intestine that were higher in the HuNoV-HS66 orally inoculated pigs. After viral challenge of the Gn pigs, the Th1 (IL-12) CSC numbers were higher in the spleen of the
VLP+mLT vaccinated pigs; however, the Th1 (IFN-γ) CSC were elicited at higher numbers in the VLP+ISCOM pigs. The Th2 (IL-4) CSC numbers were low in all three groups at PID
28; however, they were significantly higher in the VLP+ISCOM pigs compared to the
VLP+mLT pigs post-challenge in all tissues and in blood. Although the numbers of the
Th2/T-reg (IL-10) CSC were higher than those of IL-4 in the pigs that received a single dose of HuNoV-HS66, they were not significantly higher at any PID compared to controls. The
Th2/T-reg (IL-10) CSC numbers were elicited in blood at similar numbers in the VLP+mLT vaccinated pigs compared to the HuNoV-HS66 pigs at PID 28. They were significantly higher than those induced by the VLP+ISCOM pigs, but at PID 35/PCD7 the opposite was observed for the VLP+ISCOM vaccine that induced higher numbers of IL-10 CSC compared to the VLP+mLT vaccine.
277 In conclusion, both the Gn pig and calf constitute suitable animal models for the study of GII.4 HuNoV-HS66 pathogenesis and host immune responses to this strain. The
HuNoV-HS66 VLP vaccines adjuvanted with either ISCOM or mLT conferred high rates of protection against diarrhea and viral shedding, with the VLP+ISCOM inducing a more Th2 biased immune response, whereas the VLP+mLT induced a more balanced Th1/Th2 response both locally and systemically in the vaccinated Gn pigs.
278
2 IN doses of VLP+ISCOM/HS66 oral 1 oral dose of HS66 2 IN doses of VLP+mLT/HS66 oral challenge challenge Seroconversion 65 100 100 (PID 28) % IgA antibody low moderate moderate titers in serum IgG antibody low moderate (> than HS66 and VLP+mLT) moderate (> than HS66 < than VLP+ISCOM) titers in serum Coproconversion 64% 100% 100% (PID 28) IgA antibody moderate (> than HS66 at PID 28 and moderate (> than HS66 at PID 28 and < than low titers in IC VLP+mLT both pre- and post-challenge) VLP+ISCOM both pre-and post-challenge) IgG antibody moderate (> than HS66 at PID 28 and moderate (> than HS66 at PID 28 < than VLP+ISCOM low titers in IC VLP+mLT both pre- and post-challenge) both pre- and post-challenge) ASC numbers 279 In intestine low number of IgA and IgG High numbers of IgA and IgG ASC (10- moderate IgA and IgG ASC numbers (3-fold> HS66) (PID 28) ASC fold>HS66) In intestine >VLP+mLT pre- and post-challenge < VLP+ISCOM pre- and post-challenge (PID 35/PCD7) Systemically Spleen Low number of IgA and IgG (>HS66 and low numbers of IgA and IgG Low IgA and IgG (> HS66) (PID 28) VLP+mLT) Spleen < VLP+mLT > VLP+ISCOM (PID 35/PCD7) Low numbers of IgA ASC ( = HS66; < Blood Low numbers of IgA and IgG low IgA ( 3-fold > HS66; > VLP+ISCOM VLP+mLT pre-challenge) (PID 28) ASC low IgG (4-fold > HS66 at PID 28; > VLP+ISCOM) Low IgG ASC numbers (=HS66; < VLP+mLT Blood Low IgG ASC numbers (=HS66; < VLP+mLT ) low IgG (4-fold > HS66 at PID 28; > VLP+ISCOM (PID 35/PCD7)
Table 5.1. Comparison between antibody titers in serum and intestinal contents and antibody-secreting cell numbers locally and systemically in HS66-orally
inoculated pigs and pigs vaccinated with 1 oral and 2 IN doses of either vaccination regimen (VLP+ISCOM and VLP+mLT).
1 oral dose of HS66 2 IN doses of VLP+ISCOM/HS66 oral challenge 2 IN doses of VLP+mLT/HS66 oral challenge
Cytokine -IL-6= low early (PID 4) -IFN-γ= moderate (10-fold>HS66; = VLP+mLT -IFN-γ = moderate (10-fold >HS66; = concentrations -IL-12= moderate (constitutively) at PCD7) VLP+ISCOM at PCD7) Serum -IFN-γ = low early (PID 2) -IL-4=low early (PID 2,4,6,8) -IL-10= low early (PID 4,6,8) IC IFN-α= early (PID 2, 6) and late (PID21) Not sign -IFN-γ= high, late (6-fold > VLP+ISCOM at IL-12= late (PID 28) PCD7) CSC numbers -IL-6= low, early (PID4) -IL-12= low (PID21) In intestine -IFN-γ = low, early (PID2) and late (PID28) (early PIDs) -IL-4= low late (PID 28) In intestine -IL-6=low -IL-6= low (>HS66) -IL-6= low (>HS66) (PID 28) -IFN-γ = low -IL-12 = low (< HS66) -IL-12 = low (< HS66; = VLP+ISCOM) -IL-4= low -IFN-γ=low to moderate (
Table 5.2. Comparison between cytokine concentrations in serum and IC and cytokine-secreting cell numbers locally and systemically in HS66 orally inoculated pigs and
in pigs vaccinated with 1 oral and 2 IN doses of either vaccination regimen (VLP+ISCOM and VLP+mLT).
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