Novel Protein and Poxvirus-Based Combinations for Simultaneous Induction of Humoral and -Mediated

This information is current as Claire L. Hutchings, Sarah C. Gilbert, Adrian V. S. Hill and of September 24, 2021. Anne C. Moore J Immunol 2005; 175:599-606; ; doi: 10.4049/jimmunol.175.1.599 http://www.jimmunol.org/content/175/1/599 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Novel Protein and Poxvirus-Based Vaccine Combinations for Simultaneous Induction of Humoral and Cell-Mediated Immunity1

Claire L. Hutchings, Sarah C. Gilbert, Adrian V. S. Hill, and Anne C. Moore2

The presence of both cell-mediated and humoral immunity is important in protection from and clearance of a number of infectious . We describe novel vaccine regimens using combinations of plasmid DNA, poxvirus and protein to induce strong Ag-specific and Ab responses simultaneously in a murine model. Intramuscular (i.m.) immunization with plasmid DNA encoding the middle Ag of hepatitis B (DNA) concurrently with a commercial hepatitis B (HBV) vaccine (Engerix-B) followed by boosting immunizations with both modified vaccinia virus Ankara (MVA) encoding the middle Ag of HBV and Engerix-B induced high levels of CD4؉ and CD8؉ T cells and high titer Ab responses to hepatitis B surface Ag (HbsAg). Substitution of Downloaded from Engerix-B with adjuvant-free rHBsAg induced similar T cell responses and greatly enhanced Ab levels. Repeated immunizations with recombinant or nonrecombinant MVA mixed with Ag induced higher titers of Abs compared with immunization with either Ag or Engerix-B further demonstrating this novel adjuvant effect of MVA. The poxviruses NYVAC, fowlpox (FP9) and ALVAC, and to a lesser extent, adenovirus, also displayed similar adjuvant properties when used in combination with rHBsAg. The use of poxviruses as an adjuvant for protein to concurrently induce Ag-specific T cells and Abs could be applied to the development of

for many diseases, including HIV and malaria, where both cell mediated and humoral immunity may be important for http://www.jimmunol.org/ protection. The Journal of Immunology, 2005, 175: 599–606.

he major aim of vaccination is to generate sufficiently sponse is detectable in individuals with acute self-limited HBV but strong immune responses of the correct nature to protect is absent in individuals with chronic (10–12). This sug- T against disease. Most vaccines that are in used in humans, gests that a cell-mediated immune response may be important in except bacillus Calmette-Gue´rin, have been developed by optimiz- eliminating virus at an early stage of infection. A vaccine that ing their capacity to induce Ab responses. Novel candidate vac- effectively induces both cellular and humoral immunity may im- cines that have been developed to date either induce immune re- prove the efficacy of a HBV vaccination. sponses that are dominated by cellular or humoral immune Recombinant DNA and viral vectors have been used for a num- by guest on September 24, 2021 responses. For example, vaccines against intracellular pathogens, ber of years in laboratory animals and in human clinical trials as such as HIV or liver stage malaria, have been designed either to vectors for the Ag(s) of interest to induce strong effector and mem- induce strong T cell responses (1–3) or to induce Ab responses ory CD4ϩ and CD8ϩ T cell responses in both the prevention and against the extracellular form of the virus or parasite (4, 5). How- treatment of disease (2, 13–16). Widely used viral vectors include ever, it is now widely believed that the generation of both cellular modified vaccinia virus Ankara (MVA) and NYVAC, both atten- and humoral responses may be important in vaccine efficacy for uated forms of vaccinia virus, the avipoxviruses FP9 and ALVAC, diverse causes of disease including , bacteria, parasites, and and also nonreplicating adenovirus (ADV) (2, 15–21). We and cancer (4, 6–8). The licensed prophylactic vaccine to hepatitis B others have previously demonstrated in a pre-erythrocytic malaria virus (HBV),3 Engerix-B, is an example of a vaccine that induces vaccine model, where induction of IFN-␥ secreting T cells corre- Ab responses. It induces protective Abs in Ͼ85% of healthy re- cipients (9) and comprises the small surface Ag, S (HBsAg), pro- lates with protection, that heterologous prime-boost immunization duced in yeast cells (Saccharomyces cerevisiae) adsorbed to the with DNA and viral vectors is protective in mice, primate and adjuvant aluminum phosphate (alum). A vigorous CD8ϩ CTL re- human challenge models (2, 13, 16, 22, 23, 50). With respect to HBV vaccines, DNA and MVA encoding the middle Ag of HBV, comprising S and Pre-S2 Ags, have been designed in this labora- Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United tory as a potential therapeutic vaccine for chronic HBV infection. Kingdom This prime-boost regimen has demonstrated potent induction of Received for publication January 30, 2004. Accepted for publication April 29, 2005. specific T cells in mice and humans and is currently in phase II The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance clinical trials (J. Schneider, personal communication). with 18 U.S.C. Section 1734 solely to indicate this fact. In this study, we examined methods of inducing strong cellular 1 This work was supported by the Wellcome Trust. A.V.S.H. is a Wellcome Trust and Ab responses in a mouse model of candidate and licensed Principal Fellow. HBV vaccines. Although this model cannot be used to assess pro- 2 Address correspondence and reprint requests to Dr. Anne Moore, Wellcome Trust tection against HBV challenge, immunogenicity of Engerix-B can Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, U.K. E-mail address: [email protected]. be tested in mice, and this clinically licensed vaccine functions as 3 Abbreviations used in this paper: HBV, hepatitis B virus; HBsAg, small hepatitis B a good standard to compare our candidate vaccines to. We report surface Ag; MVA, modified vaccinia virus Ankara; ADV, adenovirus; ADVnr, non- here that prime-boost immunization with DNA and MVA (D/M) recombinant adenovirus; CMI, cell-mediated immunity; DC, .; LN, lymph node; DLN, draining LN; FP, fowlpox; i.d., intradermal; SFC, spot-forming induces potent T cell responses but poor levels of Ab against the cells. encoded HBsAg. Conversely, repeat immunization with Engerix-B

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 600 ENHANCED IMMUNOGENICITY BY COADMINISTRATION OF POXVIRUS AND PROTEIN induced a weak T cell response but greatly increased Abs to rH- Aldrich) to each well. Optical densities at 405 nm were measured for each BsAg compared with D/M. The concurrent administration of these well. End point titers were determined at the x-axis intercept of the dilution curve, at three times the absorbance (A ) given for naive mouse vaccines induced both T cells and Abs to HBsAg. We sought to 405 diluted from 1/200 accordingly. analyze and further improve this coinduction of cell-mediated im- munity (CMI) and humoral immunity by combining the viral vec- Statistical analysis tors MVA, FP, ALVAC NYVAC, and ADV with the main com- Statistical analysis was performed using SPSS for Windows version 10. ponent of Engerix-B, rHBsAg. Here we show that poxviruses can Mann-Whitney U tests were used to determine differences between groups. enhance T cell and Ab responses to a coadministered protein while retaining strong T cell immunogenicity for their encoded recom- Results binant Ag thereby demonstrating a method of strongly inducing Immunogenicity of DNA/MVA.HBs and protein immunization both types of immunity against a target Ag. This combination vac- T cell and Ab responses induced by the prime-boost regime of cine approach may be of benefit to the development of efficacious DNA/MVA were compared with homologous protein immuniza- vaccines where this type of immunity is required to protect against tion with or without alum (Fig. 1). challenge. Immunization with DNA/MVA induced high levels of IFN-␥ producing cells against peptide encoding a dominant CD8ϩ Materials and Methods and whole rHBsAg in the spleen (Fig. 1A) and facial DLN Animals and immunizations (Fig. 1B). Immunization with HBsAg or Engerix-B induced min- Female BALB/c (H-2d) mice (BMSU, John Radcliffe Hospital, Oxford, imal peptide and rHBsAg responses in spleen. However, the pep- U.K.), 4–6 wk old, were used in all experiments. Before immunization tide-specific response induced in axial DLN when immunized s.c. Downloaded from mice were anesthetized i.p. with 1:1:2 solution of Hypnorm (Janssen-Cilag, Saunderton), Hypnovel (Midazolam; Roche) and endotoxin-free water (Sigma-Aldrich). Plasmid DNA encoding the middle Ag of HBV was generated by in- sertion of a gene fragment containing the pre-S2 and S sequences of HBV strain ayw into the polylinker cloning region of the plasmid vector pSG2.

Expression of the middle HBsAg was driven by the CMV promoter. DNA http://www.jimmunol.org/ (50 ␮g) in endotoxin-free PBS was administered i.m. bilaterally into each musculus tibialis or mixed with 5 ␮g of Ag and injected intradermally (i.d.). MVA.HBs (MVA) contains the gene fragment containing the pre-S2 and S sequences of HBV strain ayw surface Ag inserted into the thymidine kinase locus of MVA. Expression of the middle HBsAg gene is driven by the vaccinia early/late P7.5 promoter. MVA.HBs also contains the vaccinia late promoter P11 driving expression of the lacZ marker gene. The viruses MVA, FP and ADV that were nonrecombinant for HBsAg expressed only the lacZ marker gene, termed Mnr, FPnr and ADnr, respectively. Their generation has been previously described (3, 15, 24). Nonrecombinant AL- by guest on September 24, 2021 VAC (ALnr) and NYVAC (NYnr) were a gift from Virogenetics (Albany, NY). All viruses were administered at 5 ϫ 106 pfu in endotoxin-free PBS or mixed with 5 ␮g of rHBsAg and injected i.d. bilaterally. Recombinant small surface Ag of HBV (rHBsAg), ayw subtype, was manufactured by BiosPacific in S. cerevisiae. When administered alone, rHBsAg was prepared in endotoxin-free PBS and 5 ␮g was administered s.c. into the scruff of the neck or i.d. bilaterally into the ears. Engerix-B is a commercially available vaccine containing recombinant small surface Ag (20 ␮g/ml HBsAg) produced in S. cerevisiae adsorbed to alum (GSK); 5 ␮g was administered (s.c.) into the scruff of the neck. Ex vivo IFN-␥ ELISPOT Cells from spleens were assayed for IFN-␥ production as described pre- viously (3). Axial lymph nodes (LN), which drain the s.c injection site, or facial LN, which drain the face and ears, were used to assess draining LN (DLN) responses. Axial or facial DLN were pooled within groups. Cells were stimulated for 18–20 h with 1 ␮g/ml Ld-restricted peptide IPQSLD- SWWTSL (Invitrogen Life Technologies) or 5 ␮g/ml rHBsAg. Spots were counted using an ELISPOT counter (AID) and are represented as spot- forming cells (SFC) per million ϮSEM. Ab responses Serum was collected from tail vein blood samples. Blood was allowed to clot overnight at 4°C then centrifuged at 12,000 rpm for 3 min and the FIGURE 1. Comparison of prime-boost and protein immunization. serum collected and stored at Ϫ20°C. Individual mouse serum was ana- Mice were primed with DNA i.m. (D), Engerix-B s.c. (E), rHBsAg i.d. or lyzed for anti-rHBsAg Abs by an indirect ELISA. Briefly, 96-well plates s.c. (Ag) or nothing (Nil) and boosted 2 weeks later with MVA i.d. (M), E (Maxisorp; Nalge Nunc International) were coated with 500 ng/ml rHBsAg or Ag. Two weeks after boost, cells from spleen (A) and DLN (B) were diluted in carbonate-bicarbonate buffer and incubated overnight at 4°C. assayed for IFN-␥ secretion by ELISpot. Columns represent the mean num- Plates were washed in PBS containing 0.05% Tween 20 (PBS/T) then ber of IFN-␥ SFC per million splenocytes ϮSEM (A). Cells from DLN blocked with 10% skim milk powder in PBS/T for1hat37°C. Sera diluted were pooled from individual mice in each group. Peptide or protein re- to 1/200 in PBS/T was added in duplicate wells and serially diluted. Fol- B lowing1hofincubation at 37°C, bound Abs were detected using alkaline- sponses in DLN cells ( ) are representative of several experiments. Serum phosphatase-conjugated goat anti-mouse IgG Ab (Sigma-Aldrich) or bi- levels of Abs against rHBsAg were measured by ELISA 10–13 days after ,ءءء ;p Ͻ 0.01 ,ءء .otin-conjugated anti-mouse IgG1 or IgG2a Ab (BD Pharmingen) followed boosting (C). Columns represent endpoint titer ϮSEM by incubation with ExtrAvidin (Sigma-Aldrich) for analysis. Plates p Յ 0.001 compared with mice that were immunized twice with Engerix-B were developed by adding p-nitrophenyl phosphate substrate (Sigma- (E/E). The Journal of Immunology 601 with Engerix-B or Ag was equivalent to the response induced by DNA/MVA in facial DLN. Conversely, DNA/MVA or a single immunization of Engerix-B induced very low levels of Abs against rHBsAg compared with repeat immunization with rHBsAg or En- gerix-B (Fig. 1C). These results show that prime-boost DNA/ MVA induces a potent IFN-␥ response whereas two Engerix-B immunizations induce high levels of Ab to rHBsAg. Concurrent induction of CMI and humoral immunity We assessed the concurrent induction of T cell and Ab responses by coadministration of our optimal CTL-inducing vaccine, DNA/ MVA with our strongest Ab-inducing vaccine, Engerix-B (labeled D,E/M,E). We also combined DNA/MVA with rHBsAg to exam- ine responses in the absence of alum (labeled D,Ag/MϩAg). Coadministration of these vaccines induced high levels of T cells and Abs to rHBsAg concurrently (Fig. 2). Priming with DNA and Engerix-B or rHBsAg then boosting with MVA and either Engerix-B or rHBsAg both induced statistically similar levels of peptide and rHBsAg-specific T cells compared with DNA/MVA in Downloaded from the spleens (Fig. 2A). Peptide responses in facial DLN were in- creased in groups receiving DNA/MVA with Engerix-B or rHB- sAg (Fig. 2B). Interestingly, Ab levels were reduced in the DNA/MVA with Engerix-B (D,E/M,E) group compared with repeat immunization

with Engerix-B (E/E). However, DNA/MVA with rHBsAg (D,Ag/ http://www.jimmunol.org/ MϩAg) significantly increased Abs compared with repeat En- gerix-B immunization (Fig. 2C). We demonstrate here an adjuvant effect of recombinant MVA on a coadministered protein and describe a method of vaccination whereby potent cellular and humoral immunity can be induced concurrently. Induction of Abs and T cells by homologous immunization with

MVA and rHBsAg by guest on September 24, 2021 FIGURE 2. Combination of prime-boost and protein immunizations in- Repeated immunization with the same vaccine may improve effi- duces potent CMI and humoral immunity. Mice were primed with DNA cacy in the field. We therefore aimed to induce T cells and Abs i.m. and boosted with MVA.HBsAg i.d. (D/M), or immunized twice with simultaneously using one formulation via a clinically relevant Engerix-B s.c. (E/E), or primed with DNA, i.m., and Engerix-B s.c. and route (Fig. 3). Coadministration of MVA and rHBsAg twice boosted with MVA.HBsAg, i.d., and Engerix-B s.c. (D,E/M,E), or primed (MϩAg/MϩAg) induced a high frequency of CD8ϩ T cells in with DNA, i.m., and rHBsAg i.d.(Ag) and boosted with MVA.HBsAg ϩ spleen (Fig. 3A) and DLN (Fig. 3B). Immunizing with nonrecom- mixed with Ag, i.d. (D, Ag/M Ag). Two weeks after boost, cells from spleen (A) and DLN (B) were assayed for IFN-␥ secretion by ELISpot. binant MVA (MVAnr) and rHBsAg induced weak T cell responses Columns represent the mean number of IFN-␥ SFC per million splenocytes in the spleen (Fig. 3A) although comparatively stronger responses ϮSEM (A). Cells from DLN were pooled from individual mice in each in DLN (Fig. 3B). Priming with a mixture of DNA and rHBsAg group. Peptide or protein responses in DLN cells (B) are representative of followed by boosting with MVA and rHBsAg (DϩAg/MϩAg) several experiments. Serum levels of Abs against rHBsAg were measured induced higher levels of peptide and rHBsAg specific T cells in by ELISA 10–13 days after boosting (C). Columns represent endpoint p Յ 0.001 compared with ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .spleen and DLN compared with repeat MVA and rHBsAg titer Ϯ SEM (MϩAg/MϩAg) immunization (Fig. 3A). mice that were immunized twice with Engerix-B (E/E). Immunization with MVA and rHBsAg induced a higher titer of Abs to rHBsAg compared with Engerix-B (Fig. 3C). Interestingly, priming with a mixture of DNA and rHBsAg followed by boosting some cases, Ab responses compared with homologous immuniza- with MVA and rHBsAg (DϩAg/MϩAg) or repeat MVAnr and tion (15, 16). To determine whether heterologous immunization rHBsAg immunization also increased Ab levels compared with could further increase Ab and T cell responses in our system, mice Engerix-B (Fig. 3C). were primed with DNA i.m. and a mixture of rHBsAg with either This demonstrates that two immunizations with rHBsAg mixed nonrecombinant FP (D, FPnrϩAg) or ADV (D, ADVnrϩAg) i.d. with MVA is a potent stimulator of T cells and Abs. However, All groups were boosted with recombinant MVA and rHBsAg i.d. intracellular expression of the protein, provided by recombinant (MϩAg) (Fig. 4). Intracellular Ag was found to be essential for MVA or DNA, is required for induction of specific T cells in the high cellular responses (Fig. 3A). DNA was included in the prime spleen. to provide an intracellular Ag delivery system for HBsAg in the FPnr and ADVnr groups. Heterologous immunization with FP and ADV Adding FPnr to the DNA and rHBsAg regimen did not further Lower T cell levels induced by homologous MVA immunization improve the induction of T cell responses (Fig. 4A). However, may be due to anti-MVA immune response (25). We have previ- these strong class I responses were reduced when DNA was re- ously demonstrated that heterologous priming with FP or ADV moved from the prime (Fig. 4A). In contrast, priming with and boosting with MVA induces strong cellular immunity and, in AdnrϩAg i.d. in the presence or absence of DNA i.m. induced 602 ENHANCED IMMUNOGENICITY BY COADMINISTRATION OF POXVIRUS AND PROTEIN Downloaded from http://www.jimmunol.org/

FIGURE 4. Nonrecombinant FP but not ADV is a potent stimulator of

T cells and Abs when coadministered with rHBsAg. Mice were primed by guest on September 24, 2021 FIGURE 3. Homologous immunization of recombinant MVA and rH- with DNA i.m., and rHBsAg i.d. (D,Ag), or with DNA i.m. and FPnr and BsAg induces specific T cells and Abs. Mice were primed with DNA (D) Ag mixed i.d. (D, FPnrϩAg), FPnr mixed with Ag i.d. (FPnrϩAg), DNA mixed with rHBsAg i.d. (Ag) and boosted with MVA.HBs (M) mixed with i.m. with ADVnr and Ag mixed i.d. (D, ADnrϩAg), or ADVnr mixed with Ag i.d. (DϩAg/MϩAg), or immunized twice with recombinant or nonre- Ag i.d. (ADVnrϩAg). All animals were boosted 2 weeks later with combinant MVA mixed with Ag, i.d. (MϩAg/MϩAg and MnrϩAg/ MVA.HBs mixed with Ag i.d. (MϩAg). Two weeks after the boost, cells MnrϩAg, respectively) or twice with Engerix-B s.c. (E/E). Two weeks from spleen (A) and DLN (B) were assayed for IFN-␥ secretion by ELIS- after boost, cells from spleen (A) and DLN (B) were assayed for IFN-␥ pot. Columns represent the mean number of IFN-␥ SFC per million spleno- secretion by ELISpot. Columns represent the mean number of IFN-␥ SFC cytes Ϯ SEM (A). Cells from DLN were pooled from individual mice in per million splenocytes Ϯ SEM (A). Cells from DLN were pooled from in- each group. Peptide or protein responses in DLN cells (B) are representa- dividual mice in each group. Peptide or protein responses in DLN cells (B) are tive of several experiments. Serum levels of Abs against rHBsAg were representative of several experiments. Serum levels of Abs against rHBsAg measured by ELISA 10–13 days after boosting (C). Columns represent were measured by ELISA 10–13 days after boosting (C). Columns represent -p Ͻ 0.05 compared with mice that were immu ,ء .endpoint titer Ϯ SEM p Յ 0.001 compared ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .endpoint titer Ϯ SEM nized with D,Ag/MϩAg. with mice that were immunized twice with Engerix-B (E/E).

rHBsAg, FPnr, and recombinant MVA (Fig. 4C) provides a potent similar T cell responses suggesting that ADnr adjuvants a weak method of inducing Abs at similar levels to those induced by ho- CD8ϩ T cell response that is independent of intracellular delivery mologous immunization with recombinant MVA and rHBsAg of Ag by DNA (Fig. 4A). However, these responses were greatly (MnrϩAg/MnrϩAg) (Fig. 3) (D, FPnrϩAg/MϩAg; p ϭ 0.478, reduced compared with priming with DNA i.m and Ag i.d. (Fig. FPnrϩAg/MϩAg; p ϭ 0.794, compared with MϩAg/MϩAg). 4A). All regimens induced strong peptide responses in DLN, al- This demonstrates that FPnr exhibits similar adjuvant properties to though regimens that included a DNA prime were consistently MVA in its ability to induce high levels of Abs to rHBsAg stronger (Fig. 4B). whereas, as expected, heterologous FPnr/MVA immunization with Including FPnr in the DNA and Ag prime (D, FPnrϩAg) en- DNA and rHBsAg (i.e., D, FPnrϩAg/MVAϩAg) (Fig. 4A) in- ϩ hanced Ab levels compared with DNA and rHBsAg alone (Fig. duces stronger CD8 responses compared with homologous 4C). Interestingly, priming with FPnrϩAg i.d. without DNA also MVAϩrHBsAg immunization (Fig. 3A). induced an Ab response that was not significantly different to im- munizing with D, FPnrϩAg/MϩAg, demonstrating that the en- Homologous immunization of HBsAg mixed with hanced Ab response is independent of intracellular Ag expression nonrecombinant MVA, NYVAC, ALVAC, FP or ADV at the time of priming with this vaccine. Nonrecombinant ADV, We next examined whether the adjuvanting capacity of MVA in a with or without DNA, failed to increase Ab responses when com- homologous prime-boost setting could be extended to other pox- bined with rHBsAg (Fig. 4C). Heterologous immunization with viruses or to other viruses. We tested candidate nonrecombinant The Journal of Immunology 603 poxviruses, namely, FP, NYVAC (NYnr) and ALVAC (ALnr) and recombinant MVA whereas the avipox virus, ALVACnr and re- nonrecombinant ADV (ADVnr) in combination with soluble rH- combinant MVA elicited the highest levels of anti-HBsAg Abs. BsAg (Fig. 5) Addition of each of these viruses to rHBsAg in- Homologous immunization with recombinant and nonrecombi- creased cellular responses compared with repeat immunization nant poxvirus mixed with Ag is an efficient method for inducing with rHBsAg (Fig. 5A). The most significant increase in T cell potent Ab levels against the coadministered protein and, surpris- responses compared with rHBsAg immunization was induced by ingly, the nonrecombinant poxviruses and ADV vector also in- recombinant MVA. However, nonrecombinant ALVAC, MVA, creases the T cell response induced by the coadministered protein. FP, NYVAC and ADV also significantly increased T cell re- sponses compared with rHBsAg immunization. All viruses in- duced potent responses to peptide in facial DLN although inter- Poxviruses modulate IgG subclasses estingly the most potent responses were induced by ALVACnr The ratio of isotype subclasses IgG1 and IgG2a gives an indication (Fig. 5B). of Th2 or Th1 bias of humoral responses, respectively. The effect All poxviruses enhanced the induction of potent Abs against of DNA, poxviruses, and ADV on IgG subclass division was de- rHBsAg whereas ADVnr failed to increase Ab responses above the termined (Fig. 6). level of repeat rHBsAg immunization (Fig. 5C). The attenuated Engerix-B predominantly induced IgG1 Abs which is indicative vaccinia virus, NYVACnr, induced similar Ab responses to non- of a Th2 biased immune response. This is consistent with the poor IFN-␥ production by peptide and rHBsAg stimulated splenocytes from Engerix-B immunized animals (Fig. 1A). Alum inhibited the induction of IgG2a when DNA/MVA was combined with recom- Downloaded from binant HBsAg, as no IgG2a was induced when DNA/MVA was used in combination with Engerix-B (D,E/M,E) compared with soluble Ag (D,Ag/MϩAg). In these groups, the level of IgG1 was not changed by the inclusion of DNA/MVA. Adding FPnr to this combination of DNA and Ag and boosting with MVA and Ag (D,FPnrϩAg/MϩAg) significantly increased the IgG1 and IgG2a http://www.jimmunol.org/ endpoint titers demonstrating that FP adjuvanted the prime by DNA and Ag. Removing the source of intracellular Ag, by omit- ting the DNA in this mix (FnrϩAg/MϩAg), resulted in signifi- cantly decreased IgG1 and IgG2a (D,FPnrϩAg/MϩAg compared with FnrϩAg/MϩAg; p Ͻ 0.05 for both IgG1 and IgG2a). This effect of intracellular Ag was also observed when nonrecombinant MVA was used instead of HBsAg expressing MVA in a homol- ϩ ϩ ogous prime-boost regimen (Mnr Ag/Mnr Ag). However, prim- by guest on September 24, 2021 ing with a mixture of rHBsAg and recombinant MVA or nonre- combinant FP, MVA, ALVAC, or NYVAC with or without DNA consistently increased levels of IgG2a compared with two immu- nizations with Engerix-B or Ag alone. Combination of rHBsAg with nonrecombinant MVA, NYVAC, or ALVAC, induced a 1:1 ratio of IgG1:IgG2a. In contrast, two immunizations with ADVnr and rHBsAg induced weak IgG1 and undetectable IgG2a levels compared with immunization with rHBsAg alone. Heterologous immunization, using FPnr in the prime and MVA in the boost (FPnrϩAg/MϩAg) induced higher IgG1 and IgG2a Abs com- pared with two immunizations with FPnrϩAg. The increased lev- els of IgG2a by including poxviruses with Ag indicates a Th1 biased humoral response, whereas priming with combinations in- cluding Engerix-B lead to a higher ratio of IgG1 and therefore Th2 bias. Nonrecombinant MVA, ALVAC and NYVAC increase the IgG2a response. These results indicated that equal Th1 and Th2 or biased responses can be primed to the same Ag depending on the co-delivered viral vector. FIGURE 5. Poxviruses enhance cellular and humoral immunity to co- administered rHBsAg. Mice were immunized i.d. at week 0 and 2 with rHBsAg (Ag) mixed with MVA.HBs (M/M) or nonrecombinant MVA (Mnr/Mnr), NYVAC (NYnr/NYnr), ALVAC (ALnr/ALnr), FP (FPnr/ Discussion FPnr), and ADV (ADVnr/ADVnr) or twice with Ag i.d. (Ag/Ag) or En- The presence of both CMI and humoral immunity is important in gerix-B s.c. (E/E). Two weeks after boost, cells from spleen (A) and DLN protection from and clearance of a number of pathogens (4, 8). We ␥ (B) were assayed for IFN- secretion by ELISpot. Columns represent the sought to improve the co-induction of T cell and Abs to HBsAg by ␥ Ϯ mean number of IFN- SFC per million splenocytes SEM (A). Cells using the licensed prophylactic hepatitis B vaccine, Engerix-B, or from DLN were pooled from individual mice in each group. Peptide or rHBsAg combined with DNA and viral vectors. The results here protein responses in DLN cells (B) are representative of several experi- ments. Serum levels of Abs against rHBsAg were measured by ELISA describe simultaneous induction of strong CMI and humoral im- 10–13 days after boosting (C). Columns represent endpoint titer Ϯ SEM. munity using recombinant and nonrecombinant poxviruses to ad- p Յ 0.001 compared with mice that were juvant a coadministered protein, thereby demonstrating a novel ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء immunized twice with rHBsAg i.d. method for the induction of potent T cell and Abs against a target 604 ENHANCED IMMUNOGENICITY BY COADMINISTRATION OF POXVIRUS AND PROTEIN

FIGURE 6. Serum IgG subclass responses. Mice were primed with DNA i.m and boosted with MVA.HBs i.d. (D/M), or twice with Engerix-B s.c. (E/E) or rHBsAg i.d. (Ag/Ag), or with DNA, i.m., and Engerix-B s.c. boosted with MVA.HBsAg, i.d and Engerix-B s.c. (D,E/M,E) or primed with DNA i.m., and rHBsAgi.d. and boosted with a mixture of MVA.HBsAg and rHBsAg, i.d. (D,Ag/MϩAg) or DNA i.m. with FPnr and Ag mixed i.d. and boosted with a mixture of MVA.HBs and rHBsAg, i.d (D, FPnrϩAg/MϩAg), or primed with FPnr and Ag mixed i.d and boosted with MVA.HBs and rHBsAg, i.d. (FPnrϩAg/MϩAg). Remaining groups received two immunizations of rHBsAg (Ag) mixed with recombinant MVA (MϩAg/MϩAg) or nonrecombinant MVA (MnrϩAg/MnrϩAg), NYVAC (NYnrϩAg/NYnrϩAg), FP (FPnrϩAg/FPnrϩAg), ALVAC (ALnrϩAg/ALnrϩAg), ADV (ADVnrϩAg/ ADnrϩAg). IgG1 and IgG2a subclasses of Abs against rHBsAg were measured by ELISA 10–13 days after boosting. Columns represent endpoint titer Ϯ .(p Ͻ 0.05 compared with mice that were immunized twice with Engerix-B (E/E ,ء .SEM Downloaded from

Ag. We have further demonstrated that responses can also be in- make them less effective from a practical viewpoint. We therefore duced by homologous immunization, providing a cheaper and eas- sought to simplify our vaccine regimen while still using the adju- ier method of manufacture and administration compared with het- vant capacity of recombinant MVA through homologous immu- erologous immunization. Prime-boost immunization with DNA/ nization regimens. Potent levels of T cells and Abs were induced

MVA elicited a high frequency of T cells but a minimal Ab by repeat immunization with a mixture of MVA and Ag. When http://www.jimmunol.org/ response, whereas two immunizations with Engerix-B induced compared with our initial combination vaccines used in heterolo- specific Abs to rHBsAg but poor CMI. Interestingly, combining gous prime-boost regimes, there was some reduction in T cell re- these vaccines in a concurrent prime-boost regimen induced high sponses, although a significant increase in specific Abs was elicited levels of specific T cells and Abs simultaneously. The levels of compared with our previous best regimen for inducing Abs, DNA/ peptide and HbsAg-specific T cells were maintained in the spleen MVA, and rHBsAg. Nonrecombinant MVA also demonstrated the whereas responses to peptide were increased in DLN. In all cases, capacity to enhance Ab levels to rHBsAg; however, optimal Ab T cell responses in the spleen were higher than those observed in production was induced by homologous immunization when MVA the DLN. The enhancement of immune responses in DLN by all encoded the coadministered Ag. The viral vector MVA is therefore regimens containing poxviruses or ADVnr may provide important adjuvanting responses but intracellular production of Ag is by guest on September 24, 2021 information on the strength, breadth, and longevity of the vaccine required to enhance induction of CD8ϩ T cells and to a lesser (26). T cells stimulated with the chosen peptide represent a dom- degree, Abs. MVA may achieve this adjuvant capability through inant CD8ϩ population (27, 28) whereas rHBsAg stimulation char- induction of TNF-␣ and IL-6 (25) both of which have been shown acterized total IFN-␥ production by both CD4ϩ and CD8ϩ T cells to support survival (30). It is unclear exactly how although the measured levels under-represent the total T cell re- intracellular Ag expression is able to enhance Ab responses, al- sponse probably due to limited processing of the protein into pep- though it is possible that HBsAg may be secreted from the infected tides. We verified the induction of CD4ϩ and CD8ϩ cells by in- cell (31). Alternatively, endogenous production of Ag may allow it tracellular staining (data not shown). Our initial to access an alternative route of processing and presentation, such investigations demonstrated that a strong cellular and Ab response as using nascent as well as recycling MHC class II molecules, can be achieved simultaneously. The potent cellular responses in- which may enhance CD4ϩ T cell responses. Indeed, T cell re- duced by the combination regimen were abrogated if MVA and sponses to whole Ag in vitro were increased in all cases when Ag Engerix-B were coadministered subcutaneously. This reduction in was delivered by DNA or a vectored vaccine, suggesting that T cell responses correlates with the finding that priming with DNA CD4ϩ T cell responses were induced. These CD4ϩ Th cells may i.m. followed by boosting with MVA s.c. also failed to induce high provide B cell help resulting in increased Ab responses. Immuni- levels of CTL (data not shown) and s.c. administration was shown zation with recombinant MVA leads to intracellular production of previously to be a poor route for T cell induction by recombinant HBsAg and presentation in MHC class I molecules. Nonrecombi- MVA (3). The effect of alum on MVA is unknown. Previous stud- nant MVA insufficiently stimulated class I processing of the co- ies have shown that the presence of alum can inhibit CTL induc- administered exogenous protein, hence a reduced CD8ϩ T cell tion to particulate Ags (29) and may also impair the capacity of response in this regimen. However, we demonstrate the adjuvant MVA to induce a T cell response. We therefore substituted En- capabilities of other nonrecombinant poxviruses, including AL- gerix-B with rHBsAg to avoid interference by alum. Interestingly, VAC, NYVAC, and FP, for Ab induction. Boudet et al. (19) dem- combining DNA/MVA with rHBsAg induced similar T cell re- onstrated that cellular infiltration triggered by the presence of AL- sponses compared with DNA/MVA with Engerix-B. Mixing and VAC enhanced the humoral immune response to coadministered coadministration of DNA/MVA and rHBsAg (D, Ag/MϩAg) HIV gp160 but CD8ϩ and other T cell responses were not mea- clearly increased Ab responses, in contrast to DNA/MVA and En- sured. MVA, NYVAC, and FP9 are all poxviruses and MVA has gerix-B immunization suggesting that MVA is adjuvanting rHB- also been shown to induce an inflammatory response in vitro (32). sAg but can only do so effectively in the absence of alum. Evidence suggests that viral factors and/or interaction of virus with Initial experiments indicated that combination vaccines could the host are important mediators of cellular immunity to HBsAg induce potent cellular and humoral responses but were compli- (33). In contrast to this study (33), the poxviruses we used are cated by their varied components and delivery sites which would nonreplicative in mammalian cells (34) but are still capable of The Journal of Immunology 605 inducing CMI and Abs to a coadministered protein. This suggests ministration of ADVnr in a homologous prime-boost regimen in- that the interaction between host and virus is sufficient to enhance creased the T cell response to a similar extent to repeated use of immunogenicity, most likely through affecting some form of in- some poxviruses but this strategy did not enhance Ab responses nate immunity. Viruses induce cellular infiltration and induction of above those levels observed by immunization with Ag alone. It is such as type I IFNs, TNF-␣, and IL-6 (19, 25) probably known that ADVnr induces strong anti-vector responses against through TLR-mediated signaling (35), which may lead to in- itself and may therefore be unsuitable for use in homologous im- creased uptake and presentation of encoded and delivered Ag. Pre- munization strategies (20, 21). The differential adjuvant effect of vious studies suggest that dendritic cells (DC) take up Ag or are ADV on cellular vs humoral immunity may be related to the in- infected by virus in the periphery before homing to the DLN where duction of a qualitatively different anti-vector immune response they present to T and B cells (36–39). MVA has also been shown after the priming immunization with ADV compared with poxvi- to affect DCs in vivo by inducing their activation and maturation ruses, which effects the capacity of ADV to boost humoral immu- (40). It has previously been shown that inoculation of mice with nity compared with cellular immune responses. Alternatively, it influenza and simultaneous immunization with protein induces DC may be related to different cytokines induced by each of these maturation, which was associated with specific immunity to the viruses, which then exert varied effects on humoral and cellular coadministered protein (41). The viruses we have used may be immunity. inducing DC maturation leading to enhanced uptake and presen- The example of HBV prevention provides a good illustration of tation of coadministered rHBsAg. These Ag-loaded DC may then how our strategies might be of clinical value. Approximately 5% migrate to the DLN where they stimulate naive and memory T of HBV-vaccinated recipients do not generate protective Ab levels cells, a possible explanation for why we see such potent responses (9). In our regimens, we demonstrate not only an increase in spe- Downloaded from following immunization with the viral vectors. cific Abs but also a boost in CD4ϩ T cells, which may in part be It is well established that recombinant poxviruses induce in vivo responsible for this amplification of Ab levels. Increasing the num- expansion of pre-primed CD8ϩ T cells (2, 17, 42) but are also ber of CD4ϩ T cells may overcome the problem of nonresponders immunologically potent when different viruses encoding the same to this vaccine and possibly to other vaccines. Clinical trials of Ag are used in prime and boost (15, 16, 43, 44). We recently investigational vaccines involve the use of recombinant viruses

reported that heterologous prime-boost immunization with FP and and protein that are generally used separately at different time http://www.jimmunol.org/ MVA, both encoding the same malarial Ag, induced greater num- points (48, 49). We demonstrate that combination vaccines, bers of specific CD8ϩ T cells and protection from challenge when whereby the virus and protein-based vaccines are concurrently ad- compared with homologous immunization with either vector (16). ministered is an alternative strategy that induces enhanced immu- To determine whether heterologous prime-boost could improve nogenicity. The mouse model is unsuitable as a HBV challenge immunogenicity in our system, animals were primed with DNA, model, therefore we cannot directly demonstrate that coadminis- rHBsAg, and nonrecombinant FP9 then boosted with MVA and tering viruses and recombinant HBsAg results in improved pro- rHBsAg. DNA was included in the prime to provide intracellular tection against virus. However, we believe that the significant in- expression of HBsAg and for enhancing T cell responses. Inclu- crease in the magnitude and robustness of the Ab and T cell sion of FP9 at prime increased T cell responses whereas similar Ab responses induced by these combination vaccines may result in by guest on September 24, 2021 levels were maintained when compared with homologous MVA improved protection against pathogen challenge. We have recently and rHBsAg immunization. This result supported heterologous im- successfully applied this strategy to a malaria challenge model in munization as a more effective strategy than homologous immu- mice (C. Hutchings, A. Birkett, A. Moore, and A. Hill, manuscript nization for T cell induction. Kent et al. (45) demonstrated that in preparation). Ag-specific T cells primed by a single DNA immunization were Here we describe a novel vaccine regimen capable of inducing subsequently boosted with a non-FP9 strain of recombinant FP CD4ϩ and CD8ϩ T cells and high levels of specific Abs concur- virus, but this FP boost resulted in reduced Abs. In our study, rently. We have demonstrated that combination and route of ad- including nonrecombinant FP9 in the prime with rHBsAg and ministration of the vaccine components is critical for inducing op- DNA immunization likewise primed Ab and cellular responses, timal cellular and humoral responses to the encoded and both of which were further amplified following boosting with exogenous Ag. We used a well-described subunit protein and re- MVA and rHBsAg. combinant viral vectors, both acceptable for clinical use, to en- Recombinant ADV is another commonly used vector for induc- hance immune responses. These results may have important im- ing strong responses to encoded Ags (15, 43, 46, 47). Priming with plications for the rational design of future vaccination strategies. ADV expressing a Plasmodium yoelii Ag and boosting with VV encoding the same Ag induced specific T cells and Abs and pro- Disclosures vided complete protection from P. yoelii challenge in mice (43). In The authors have no financial conflict of interest. this study, priming with DNA and nonrecombinant ADV mixed with rHBsAg followed by boosting with MVA and rHBsAg re- References sulted in a weaker peptide-specific T cell response, but similar Ab 1. Hanke, T., A. J. McMichael, M. Mwau, E. G. Wee, I. Ceberej, S. Patel, J. Sutton, levels, compared with coimmunization of DNA/MVA and rHB- M. Tomlinson, and R. V. Samuel. 2002. Development of a DNA-MVA/HIVA vaccine for Kenya. Vaccine 20: 1995–1998. sAg. In contrast to poxviruses, the adjuvant effect of ADV was 2. Moore, A. C., and A. V. Hill. 2004. 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