An Age-Old Paradigm Challenged: Old Baboons Generate Vigorous Humoral Immune Responses to LcrV, A Plague This information is current as of September 27, 2021. Sue Stacy, Amanda Pasquali, Valerie L. Sexton, Angelene M. Cantwell, Ellen Kraig and Peter H. Dube J Immunol 2008; 181:109-115; ; doi: 10.4049/jimmunol.181.1.109 http://www.jimmunol.org/content/181/1/109 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

An Age-Old Paradigm Challenged: Old Baboons Generate Vigorous Humoral Immune Responses to LcrV, A Plague Antigen1

Sue Stacy,*‡ Amanda Pasquali,* Valerie L. Sexton,† Angelene M. Cantwell,† Ellen Kraig,2*‡ and Peter H. Dube†‡

Immune senescence in the elderly results in decreased immunity with a concomitant increase in susceptibility to and diminished efficacy of vaccination. Nonhuman primate models have proven critical for testing of vaccines and therapeutics in the general population, but a model using old has not been established. Toward that end, immunity to LcrV, a protective Ag from , was tested in young and old baboons. Surprisingly, there was no age-associated loss in immune competence;

LcrV elicited high-titer, protective Ab responses in the older individuals. The primary responses in the younger baboons were Downloaded from lower, but they did show boosting upon secondary immunization to the levels achieved in the old animals. The LcrV Ag was also tested in mice and, as expected, age-associated loss of immunity was seen; older animals responded with lower-titer Abs and, as a result, were more susceptible to Yersinia challenge. Thus, although age-related loss in immune function has been observed in humans, rodents, and some nonhuman primates, baboons appear to be unusual; they age without losing immune competence. The Journal of Immunology, 2008, 181: 109–115. http://www.jimmunol.org/ lderly individuals show diminished immune responses, (17–20). Similarly, the age-associated loss of immunity can be making them significantly more susceptible to overcome by giving multiple immunizations or higher doses of the E and cancer (reviewed in Ref. 1–4). In addition, vaccina- Ag. This further substantiates the imperative to test vaccine pro- tion protocols are typically less efficacious in the elderly and al- tocols for their effectiveness in both old and young subjects. though higher doses of immunogen may enhance the response, it Although many vaccines are first tested in rodents, this may not is typically still lower than the one elicited in younger individuals be ideal for protection studies because mice are resistant to many (5–8). Deficits in the ability of older subjects to generate immune human (like HIV) due to sequence differences in their responses, particularly to “new” Ags that they have not previously cellular receptors. Thus, many vaccine protocols for use in humans encountered, have been widely reported. In contrast, immune have been tested in nonhuman primate (NHP)3 models (21–26). by guest on September 27, 2021 memory to Ags encountered in one’s youth does survive aging and However, the vast majority of these studies have been undertaken can be recalled in old age (9–10). Given the current demographic in young or middle-aged NHPs and none of these primate models composition in the U.S., the numbers of aging individuals will has been validated for use in testing vaccines for efficacy in older continue to grow and they already comprise a significant “at risk” individuals. Thus, in the current study, the ability of young and old population. Thus, it is critically important to develop and test pro- NHPs to respond to a “new” Ag has been assessed. We chose to tocols for enhancing immunity particularly to “new” Ags in old, as focus on the baboon, Papio hamadryas, for several reasons. It is an well as in young, individuals. excellent primate model system due both to its close genetic re- Most of the research into the effects of aging on immunity has latedness (Ϸ96% DNA homology) and the similarity of its im- incorporated rodent models and, for the most part, analogous age- mune system to humans (27). For example, unlike macaques and associated deficiencies of cellular and humoral immunity have some other monkeys, baboons resemble humans and chimpanzees been seen (11–16). For example, the ability to generate an immune in exhibiting four IgG subclasses (28). Moreover, because baboons response to a “new” Ag or epitope not previously encountered is breed well in captivity, they are more readily available than some significantly diminished in older animals. In contrast, memory im- other NHPs. Baboons are being used extensively in infectious dis- munity to Ags encountered in one’s youth appears largely intact ease and vaccine studies (21, 24–25, 27) so it will be important to assess the effects of aging on this NHP model. It has been reported that serum autoantibodies in baboons increase with age, analogous *Department of Cellular and Structural Biology, †Department of Microbiology and to humans (29), but there are no studies that assess the effects of Immunology, and ‡Barshop Center for Longevity Studies, University of Texas Health aging on humoral immunity. Fortunately for this study, the South- Science Center, San Antonio, TX 78229 west National Primate Research Center in San Antonio maintains Received for publication July 27, 2007. Accepted for publication April 22, 2008. the largest colony of baboons worldwide; it consists of Ͼ3700 The costs of publication of this article were defrayed in part by the payment of page individual animals, including a geriatric cohort. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Given that aging most dramatically affects immune responses to 1 This work was supported by funding from the Southwest National Primate Center Ags not previously encountered by the subject, it was imperative (pilot study Grant P51 RR13986), the National Institute on Aging (R03AG22675 to S.S.), and a University of Texas Health Science Center, San Antonio Presidential Research Enhancement Fund grant. 3 Abbreviations used in this paper: NHP, Nonhuman primate; TTFC, Tetanus toxoid 2 Address correspondence and reprint requests to Dr. Ellen Kraig, Cellular and Struc- fragment C. tural Biology MC 7762, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org 110 BABOON IMMUNE COMPETENCE IS MAINTAINED WITH AGE to select an immunogen that would elicit a primary response in the baboon colony. Thus, we chose LcrV, a Ag from Yersinia pestis, the causative agent of bubonic plague. Y. pestis is the most virulent bacterial currently known and in geographic ar- eas where it is endemic in rodent populations, including the south- western U.S., humans remain at risk. Any baboon that had come into contact with Y. pestis would most likely have succumbed, as the infection is typically fatal. Thus, none of the subjects used in this study were likely to have had a prior exposure to this virulent bacterium and, therefore, they should respond to LcrV as a “new” Ag. Furthermore, although there is no currently licensed plague vac- cine for use in the U.S., a new subunit vaccine, which includes FIGURE 1. Preparation of protein immunogens. The recombinant pro- LcrV as one of its components, is showing promise (30–32). By teins were prepared in a prokaryotic expression system and purified, as described in Materials and Methods. In order to demonstrate purity, they incorporating LcrV in this study, we could assess both the titer of were fractionated on a SDS polyacrylamide gel and stained with Coomas- reactive Ab produced and its ability to protect against infection. sie Blue. A duplicate gel was electrophoretically transferred and the re- This was considered a significant advantage as there is growing sulting Western blot was developed using an Ab that recognizes the vector- concern that plague may re-emerge as a significant danger to hu- encoded “his” tag. man health due to the recent identification of multidrug resistant Downloaded from strains of Y. pestis, thus, making the development of an effective vaccine a priority (33–34). Yet, in no case has a protein-based an Ab against the vector-encoded “his” tag (Qiagen) and developed using vaccine for plague been tested in older animals whose immune an alkaline phosphatase conjugated secondary Ab. responses are likely compromised. Thus, these studies will provide Baboon immunization the first data on the effects of aging on the humoral immune response The purified recombinant were absorbed to 25% alum (Vol/Vol) to LcrV in two different species, mice and baboons. Moreover, al- http://www.jimmunol.org/ and used to immunize young (21⁄2 years) and old (19–24 years) baboons though the Y. pestis LcrV Ag was chosen for this study, our findings from the pedigreed colony at the Southwest National Primate Research should be generalizable to all protein-based and subunit vaccines and Center. Each was vaccinated by i.m. injection with 100 ␮g of LcrV should also provide important insights into the use of NHP models for or the chimeric Ag, LcrV::TTFC. Sera were obtained from each animal testing vaccine efficacy in the elderly. before immunization (preimmune) and at intervals following the primary immunization (4 and 8 wk). All of the baboons received a second immu- nization with 100 ␮g of recombinant LcrV in Alum 6 mo subsequent to the Materials and Methods primary exposure. The animals were bled 2 and 6 wk after the secondary inoculation.

To produce recombinant immunogens, wild-type CO92 genomic DNA, Mouse immunizations by guest on September 27, 2021 containing the pMT and pCD1 virulence , was used as the tem- C57BL/6 mice were obtained from the NIA contract colony (Harlan caf1 lcrV plate in PCR to amplify the (F1) and . The primers used for Sprague Dawley) and were used at either 3 mo of age (young) or 19–21 mo F1 were: 5Ј-GAC GAC GAC GAC AAG GAT TTA ACT GCA AGC (for- ␮ Ј (old). The old and young mice were immunized with 10 g of LcrV, ward) and 5 -TCA CTA TCA TCA TTA TTG GTT AGA TAC GGT TAC LcrV::TTFC, or F1 Ag in alum. The mice were bled from the retro-orbital (reverse). The primers designed to amplify the LcrV coding sequence were: sinus before immunization and at intervals post primary (4 and 9 wk) and 5Ј-GAC GAC GAC GAC AAG ATG ATT AGA GCC TAC GAA CAA Ϫ Ј secondary (2.4 and 5.4 wk) exposures. The sera were frozen at 20°C for AAC CCA CAA CAT (forward) and 5 -AAG ACC TTG TGA GCA TCC subsequent analyses. TCG (reverse). In addition, each of the forward primers included sequences added at the 5Ј end to introduce an enterokinase cleavage site; this is shown Measuring Ab titers in italics in the primer sequences above. The LcrV and F1 PCR products were TA cloned into the bacterial expression vector pQE-30UA vector Direct ELISAs were developed to measure the levels of serum Abs specific (Qiagen) which provides an amino terminal histidine tag to facilitate pro- for LcrV and F1 using the recombinant proteins generated above. For as- tein purification. The chimeric Ag, LcrV::TTFC was created by subcloning sessing reactivity to TTFC, a recombinant protein lacking the “his” tag was the tetanus toxoid fragment C (TTFC) coding sequence in-frame down- obtained from commercial sources (Roche). In all cases, Immunosorb 96- stream from LcrV at the BamHI site in pQE-30; the original TTFC clone well plates (Nunc) were coated with the appropriate Ag (5 ␮g/ml for LcrV was provided by Dr. Robert Ulrich (Army Medical Research Institute of and F1, 10 ␮g/ml for TTFC). A dilution series of each baboon or murine Infectious Diseases, Frederick, MD) and is described elsewhere (35). All sera was prepared and incubated with the Ag. Specific Ab binding was three inserts were sequenced by the University of Texas Health Science detected with HRP-conjugated anti-monkey IgG (Kirkegaard & Perry Lab- Center at San Antonio DNA Core Facility to ensure that the proper se- oratories) or rabbit anti-mouse IgG (Sigma-Aldrich). The ELISAs were quence had been cloned as a functional translational fusion. developed by the addition of a chromogenic substrate ABTS and the ab- The recombinant clones were transformed into Escherichia coli BL21 sorbance at 410 nm was determined. Titers were fitted to a sigmoidal curve (DE3) pLys S and expression was induced by the addition of isopropyl (GraphPad Prism 5) and the end-point titers at 0.1 OD above background ␤-D-thiogalactoside. pellets were collected and disrupted by sonica- were determined by interpolation. tion and the lysate was cleared by centrifugation at 20,000 ϫ G for 20 min. Statistically significant differences between young and old animals were Recombinant proteins were purified from the soluble fraction using affinity determined by one-way ANOVA ( p Յ 0.05). To satisfy the assumption of chromatography on nickel chelating resin (Pharmacia) and eluted with 500 variance equivalence among treatment groups, a log base ten transforma- mM imidazole. The fractions containing recombinant protein were further tion of the data was performed. The statistical significance of differences in purified by gel filtration followed by ion exchange chromatography on a responses between the young and old animals in a given treatment group resource Q column before being eluted with 500 mM NaCl. Positive frac- was then assessed by one-way ANOVA on the transformed data. tions were pooled and the buffer was exchanged to a PBS solution (pH 7.4) ELISAs were also performed using HRP-conjugated sheep Abs specific on a gel filtration column. Contaminating endotoxin was removed with for human IgG1 (AP006), IgG2 (AP007), IgG3 (AP008), and IgG4 polymixin agarose (Sigma-Aldrich) and the protein was stored in aliquots (AP009) from The Binding Site. For this purpose, ELISA plates were at Ϫ80°C before use in immunization protocols. coated with recombinant LcrV as described above and then incubated with To demonstrate purity of the recombinant proteins, a sample of each was baboon and mouse sera diluted 1/500; this dilution was within the linear fractionated by size on an SDS-polyacrylamide gel and stained (see Fig. 1). range for total IgG. The secondary Abs were then tested in duplicate wells In addition, a duplicate gel was electrophoretically transferred to Duralon at three different dilutions (1/1000, 1/3000, and 1/9000). The plates were Membrane (Millipore) and the resulting Western blot was incubated with developed and read as described above. The Journal of Immunology 111

challenged intradermally with 119 cfu of CO92 in the ear as described (36). The were originally obtained from the Centers for Disease Control and Prevention Select Agent Distribution Activity and were grown over- night at 37°C in heart infusion broth supplemented with 0.2% xylose. The actual challenge dose delivered to the mice was determined by plating serial dilutions of the bacteria on Congo red plates and enumerating the number of colony forming units. In our hands, the ID LD-50 for CO92 is between 1 and 10 cfu (Dube, unpublished observations). The mice were monitored daily and scored for percent survival. Assessing protection from Y. pestis in immunized mice Young and old mice were immunized twice with Alum (negative control) or with one of the test immunogens in Alum, Y. pestis F1 Ag, LcrV, or a chimeric molecule LcrV::TTFC, as described above. They were then chal- lenged intradermally with 8000 cfu Y. pestis CO92. The percent survival (10 mice/group) was scored daily.

Results Primary baboon immune responses to Y. pestis LcrV are not impacted by aging

To test the effects of aging on immune responses to a protein Ag Downloaded from in a NHP model, the Y. pestis LcrV was cloned into a pro- karyotic expression vector and protein immunogen was prepared. FIGURE 2. Effects of age on the Ab response to LcrV in baboons and To demonstrate purity of the recombinant LcrV, it was subjected mice. Young and old animals were immunized twice with Y. pestis LcrV in to SDS-PAGE and Western blot analysis using an Ab against the alum and were bled at the indicated time points after the primary and vector-encoded “his” tag. As shown in Fig. 1, there was one major secondary immunizations. ELISAs were performed in duplicate with di- protein species detected and it was of the size predicted for the http://www.jimmunol.org/ luted sera on plates coated with recombinant LcrV and end-point titers “his”-tagged LcrV. The purified LcrV was then absorbed to alum, were determined using the GraphPad Prism 5 program. The data for both an adjuvant approved for use in humans, and used to immunize baboons (A) and mouse (B) are shown. The titers from young animals are young (21⁄2 years) and old (19–24 years) baboons from the pedi- Ⅺ indicated using open bars ( ) and those from old animals are indicated greed colony at the Southwest National Primate Research Center. with the filled bars (f). Statistically significant differences between young Sera were obtained from each baboon before immunization (pre- and old animals within a treatment group were determined by one-way ANOVA as described in Materials and Methods (p Յ 0.05 are indicated by immune) and at 4 and 8 wk following the primary inoculation. The titer of LcrV reactive Abs in each sample was determined by direct .((ء) an asterisk ELISA and the end-point titers were calculated using the Graph- Pad Prism 5 program; the results are shown in Fig. 2. Because by guest on September 27, 2021 Measuring the protective capacity of the baboon Ab response end-point titers could not be determined for the preimmune sera, To assess the protective capacity of the baboon sera, pools of individual all individual baboon samples were retested at one dilution point sera from a given time point were tested for the ability to protect mice from within the linear range (1/1200) to allow direct comparison across Y. pestis challenge; this is the US Public Health Service approved method samples; these data are shown in Table I. As expected, the preim- for measuring protective immune responses to this virulent pathogen (30). mune titers to LcrV were relatively low, although one of the young In brief, 0.5 ml of each pool (from a given time point) was used to pas- sively immunize female CD-1 mice (5/group). As a negative control, one baboons did have a slightly elevated level (Table I). Upon immu- group of mice was given 0.5 ml PBS and tested in parallel. One day after nization, the titers increased in all animals relative to the preim- serum transfer, the mice were sedated with avertin 0.5 mg/kg i.p. and mune levels, but, unexpectedly, the elderly baboons responded at

Table I. Ab responses of individual baboons immunized with LcrV

LcrV Titera

Time after LcrV Time after Preimmune Immunizationb Boostc

Age group Animal No. Year of Birth 0 wk 4 wk 8 wk 2 wk 6 wk

Young 19373 2003 0.01 0.25 0.09 0.56 0.24 19384 2003 0.01 0.44 0.19 0.58 0.37 19047 2003 0.08 0.28 0.14 0.59 0.37 19060 2003 0.27 0.37 0.16 0.61 0.57 mean 0.09 0.34 0.14 0.58 0.39 Old 1X4039 1981 0.02 0.45 0.46 0.61 0.47 1X4280 1982 0.01 0.56 0.41 0.53 0.37 1X4558 1983 0.02 0.22 0.18 0.35 0.25 1X4837 1985 0.03 0.64 0.50 0.62 0.53 6280 1985 0.02 0.41 0.23 0.74 0.58 6800 1986 0.02 0.30 0.17 0.58 0.30 mean 0.02 0.43 0.32 0.57 0.42

a (A410) determined by LcrV ELISA at a serum dilution in the linear range (1/1200). b The primary immunizations were given in August, 2005. c The secondary immunizations were given in February, 2006. 112 BABOON IMMUNE COMPETENCE IS MAINTAINED WITH AGE least as well as the younger animals; there was no age-associated loss in immune competence (Fig. 2A). Primary murine responses to LcrV do show an aging effect The vigorous immune responses in the old baboons immunized with LcrV were unexpected and we considered that the Ag itself, LcrV, could be skewing the results (possibly by suppressing the responses in the young animals). To address this unlikely possi- bility, young (3 mo of age) and old (19–21 mo) C57BL/6 mice were immunized with the same antigenic preparation, recombinant LcrV in alum. The mice were bled before immunization and at the prescribed intervals after exposure; the sera were then tested for anti-LcrV titers by direct ELISA. As shown in Fig. 2B, the mice responded as expected; old mice show lower titers upon LcrV immunization than do the young animals. Thus, the Ag used did not have a negative effect in young mice. It appears more likely that baboons do not undergo the expected age-associated decline in immune responsiveness that has been reported for humans and numerous animal models. Downloaded from Response to a chimeric Ag confirms lack of immunosenescence in baboons To pursue this question further, old and young animals were im- munized with a second immunogen, a chimeric molecule com-

posed of LcrV and TTFC; the purity of this immunogen was sim- http://www.jimmunol.org/ ilarly demonstrated (Fig. 1). Because they were immunized with a chimeric molecule, the baboons and mice should produce Abs to FIGURE 3. Effects of aging on the Ab responses to a chimeric Ag, each of the individual components, LcrV and TTFC. Therefore, LcrV::TTFC. Old and young animals were immunized with recombinant ELISAs were performed independently with these two Ags (Fig. chimeric LcrV::TTFC protein in alum. Sera, collected before immunization 3). In both cases, old baboons responded as well, if not better, than and at the indicated times thereafter, were tested for the presence of anti- the young animals. In mice, as expected, the pattern was reversed; LcrV Abs and also for anti-TTFC Abs using direct ELISAs. The end-point young responded better than old. titers were determined and are presented for baboons (A) and mice (B). The titers from young animals are indicated using open bars (Ⅺ) and those from

Effects of age on secondary responses in baboons and mice old animals are indicated with the filled bars (f). Statistical significance of by guest on September 27, 2021 the differences between old and young within a treatment group was as- In mice and humans, boosting of the immune response is expected sessed as described in Materials and Methods. with subsequent exposures to the Ag due to the prior activation of memory B and T cells. Because the primary immune responses in old baboons had been surprisingly vigorous, in comparison to young baboons, we asked whether memory T and B cell responses The kinetics of survival (protection) is shown for the serum had been generated. Briefly, the mice and baboons previously im- samples taken 4 wk after the primary immunization (second panel munized with LcrV were given a secondary immunization and the in Fig. 4A). As seen with the preimmune samples, the young im- titers of the resulting responses were measured by ELISA. The sec- mune sera provide little protection. However, transfer of the sera ondary responses were higher for both young and old mice, as ex- from old baboons, even after a single immunization, provided high pected (Fig. 2). In baboons, the secondary responses were boosted for level protection from Y. pestis challenge. The survival data for all the young animals, but were not enhanced over the primary responses time points taken after primary immunization are summarized (ta- for the older animals (Fig. 2 and Table I). Thus, in both cases, mem- ble on right, Fig. 4A). The protective capability of the old baboon ory immune responses were generated but again, the baboon profile sera had diminished by 8 wk after immunization. This was some- does not look like the one typically seen in mice or humans. what surprising because there were still relatively high titers at this time. However, the protection assay is more physiologically rele- Elicitation of protective humoral responses in old and young vant; it measures the functional capability of the Abs elicited. baboons The ELISA titers were also generally predictive of protection Even though the elderly baboons showed higher Ab responses to after secondary exposure. For both young and old baboons, rela- LcrV immunization when compared with the young, it was pos- tively high titers were seen within 2 wk after immunization and sible that the Abs produced by the aged animals would not be as these Abs were protective (third panel of Fig. 4A). However, as capable of neutralizing virulent bacteria. To test this possibility, seen with the primary immunization, protection declined relatively mice were passively immunized with baboon sera pooled from a quickly and was below the 80% cut-off by 6 wk postsecondary given time point and were then challenged with virulent Y. pestis. immunization (Fig. 4A, summary table). Thus, these data confirm The mice were scored daily; a baboon serum was considered pro- that the old baboons generate Abs to LcrV that are both high titer tective if at least 4/5 recipient mice (Ն80%) survived 10 days. and protective in vivo. Protection was not seen in the mice receiving young preimmune sera (0% survival, Fig. 4A), old preimmune sera (20% survival, Fig. 4A), Elicitation of protective humoral responses in old and young or PBS (0% survival, data not shown). Thus, the infectious dose de- mice livered was sufficient to cause terminal disease and the baboons did For comparison, the generation of protective responses in mice not have significant pre-existing immunity to Y. pestis. was tested directly by immunization and subsequent challenge The Journal of Immunology 113 Downloaded from http://www.jimmunol.org/

FIGURE 4. Effects of aging on the generation of protective Abs in baboons and mice. A, To assess the protective capacity of the baboon sera, female CD-1 mice (5/group) were given 0.5 ml of a pool containing equivalent amounts of individual sera taken at a given time point. As a negative control, one group of mice was given 0.5 ml PBS and tested in parallel. One day after serum transfer, the mice were challenged by intradermal injection of 119 cfu of Y. pestis CO92. They were monitored daily and scored for percent survival. The three panels on the left show the survival curves obtained for young and old baboon sera taken either before immunization (preimmune) or after primary or secondary immunization. The summary table to the right in A reports the number of animals surviving 10 days for each of the time points tested. B, Young and old mice were immunized twice with alum (negative control) or with one of the test immunogens, Y. pestis F1 Ag, LcrV, or a chimeric molecule LcrV::TTFC. They were then challenged (as described in A) with 8000 cfu Y. pestis CO92. The percent survival (10 mice/group) was scored daily and is shown. by guest on September 27, 2021 with virulent Y. pestis, following the secondary exposure. As F1. Thus, as expected, aging negatively impacts immunity in mice. shown in Fig. 4B, protection correlated with Ab titer, for the time In contrast, older baboons appeared to have a healthier immune points tested. As expected, old mice were much more susceptible system than the younger animals. to challenge, presumably due to the lower titers of anti-LcrV Abs elicited. This finding was generalizable to three different immuno- Isotype profiles of LcrV-specific Abs produced by young and gens, LcrV, LcrV::TTFC, and another plague vaccine component, old baboons There have been well documented changes in expression with aging. This could impact the class of Ab elicited in response to certain immunogens or pathogens. To assess whether age is affecting H chain switching in the primates, we determined the proportions of IgG subclass Abs obtained in response to LcrV immunization. Like humans, baboons have four subclasses of IgG and the secondary reagents sold by The Binding Site have been reported to work with either of these primate species (28). Thus, we performed an ELISA to compare the isotype profiles of young and old baboons responding to LcrV immunization. The levels of IgG3 and IgG4 were below detection in all samples (data not shown), while IgG1 and IgG2 dominated the responses (Fig. 5). There was no significant difference in the subclass distribution seen with age. Thus, these data confirm that the old baboons gen- erate Abs to LcrV that are high titer, protective in vivo, and of FIGURE 5. Effects of aging on the Ig subclass of Abs produced in re- similar Ig subclasses to those seen in younger animals. sponse to LcrV immunization of baboons. The primate sera from old (O) and young (Y) LcrV-immunized baboons were retested by ELISA using Ig Discussion subclass-specific secondary Abs from The Binding Site. For this experi- ment, the baboon sera were all used at a single dilution point (1/500) It has been generally accepted that all animals undergo immunose- previously shown to be within the linear range of the assay. The average nescence associated with a decline in the ability of older individ- Ϯ uals to mount a protective immune response to Ags not previously A410 reading SEM are shown for each of the three dilutions of secondary Ab used for IgG1 and IgG2. The levels of IgG3 and IgG4 reactive with encountered. This report demonstrating that older baboons are at LcrV were at baseline and are not shown. least as good as young animals at generating a protective humoral 114 BABOON IMMUNE COMPETENCE IS MAINTAINED WITH AGE response to LcrV challenges that age-old paradigm. We considered In humans and rodents, immunosenescence is accompanied by several factors that might have contributed to this surprising find- decreased Ab affinity, due to diminished somatic mutation in older ing. First, it was possible, although highly unlikely, that the spe- individuals (38). A decrease in affinity for Ag would be expected cific baboon ages chosen for the analysis were not appropriate. The to negatively impact its ability to function optimally in response to young baboons were 21⁄2–3 years old when first immunized, which a pathogen and may well compromise its neutralizing capabilities. approximates 71⁄2–9 year old humans. As juveniles, they would Thus, we might have expected that the anti-sera from old baboons, have attained immunological maturity, but not sexual maturity. despite their relatively high titer Ab content, would have proven This age group was chosen based on extensive baboon data dem- less efficacious in the passive transfer protection assay. However, onstrating that they would be immunologically competent (much this was not the case; the sera from old baboons, even after a single like school-age children). For example, Attanasio and colleagues immunization, were significantly more potent in protecting from Y. (29) demonstrated that the levels of serum Ig are close to adult pestis challenge than were the sera from younger individuals. levels by 1 year of age in baboons. In addition, in a West Nile virus Other aspects of the aging immune system in baboon have been vaccine study, a 3-year-old juvenile baboon was immunized and studied, with mixed results. For example, old baboons show a de- generated IgG levels as high or higher than those seen in young crease in the number of B cells and an increase in T cell numbers adults (5.5–9 years of age) (24). In fact, even fetal baboons are with age (39); this is unlike humans. However, some aspects of the capable of responding with IgG responses upon immunization with immune dysregulation seen in humans and rodents may also occur hepatitis B virus Ags (37). The older animals (19–24 years of age) in baboons because the levels of serum autoantibodies do increase were chosen to be Ն2/3 of the average live span, which would be with age (29), suggesting that tolerance mechanisms may be im- roughly equivalent to 57- to 72-year-old humans and to 24-mo-old pacted by aging. By comparing the aging human immune system, Downloaded from mice. If immunosenescence occurred equivalently in baboons, which shows profound deficiencies in immunity, to the baboon these animals should have shown a significant decrease in their Ab immune system, which is less impacted, we may gain important titers upon immunization, relative to the younger animals. Instead, insights into immunosenescence and novel mechanisms to slow they exhibited just the opposite; old baboons responded at very the process down. Importantly, given that the expected decrease in high levels to immunization. immunity to newly encountered Ags does not occur in aging ba- In mice, the decline in immune capability has been shown to be boons, other NHP models should be considered for testing vaccine http://www.jimmunol.org/ compositions and immune-based therapeutics, particularly for their a gradual one; middle-aged animals produce a humoral response use in the elderly. There have been only a few studies in NHP to that is intermediate, falling between the very low levels seen in assess the effects of aging on the immune system. Thus far, the older mice and the much higher levels produced by younger ones results have been quite mixed, but there are parallels seen to hu- (14, 17). Given that the old baboons used in this study were not mans in several of the NHP species tested. For example, thymus extremely old or geriatric, we expected detectable induced titers, involution does occur with aging in macaques (40). Moreover, but these should, if the paradigm were correct, have been lower lymphocyte proliferative responses and humoral immunity have than those achieved in younger animals. Clearly, this was not the been shown to decline with age in these monkeys, but the changes case; aging appears to have less of an effect on humoral immunity by guest on September 27, 2021 did not entirely mimic humans (41). Additional studies in rhesus in baboons than in other species examined to date. macaques have shown that CD28 expression declines with age as We next explored the possibility that the old baboons had been it does in humans but signaling and cell cycle regulation appear to previously exposed to LcrV. If so, the immunization for this study differ (42, 43). Similarly, in aging cynomolgus monkeys, an in- would have boosted an existing memory response, which would crease in double-positive (CD4ϩCD8ϩ) T cells was demonstrated potentially account for the unexpectedly high responses in the el- (44), suggesting a possible loss of thymic selection. Before selec- derly baboons. However, we consider it highly unlikely for several tion of a particular NHP for use in testing vaccines or therapeutics, reasons. First, we chose the Ag, LcrV, specifically because the the responses to standard immunization protocols should be tested animals should have been naive. Y. pestis has not been reported at in young and old individuals to assess whether they show immu- the Southwest National Primate Research Center and had any an- nosenescence, like humans, or fail to show age-associated losses in imal been infected, it would likely have died previously. More- humoral immunity, like baboons. over, due to the extreme susceptibility of NHP to Yersinia spp, it is also unlikely that these animals were exposed to the related Acknowledgments enteropathogenic species of Yersinia. Second, none of our animals We express our gratitude to Dr. Karen Rice and Sabrina Chatman for had been previously enrolled in a study involving any Ag from this providing expertise and for coordinating the nonhuman primate protocols bacterium. Third, another Ag, TTFC, as part of the chimeric mol- and procedures. We also thank Dr. William Morgan for very helpful advice ecule, also elicited high responses from the older baboons. We in the statistical analysis of these data. In addition, we are grateful to Dr. cannot unconditionally eliminate the possibility that the old ani- Robert Ulrich for providing the TTFC clone. Lastly, we express our ap- mals had been exposed to some other bacterium or Ag that elicited preciation to Dr. Philip LoVerde whose laboratory screened the Binding a response that would be cross-reactive with LcrV and/or TTFC. Site secondary Abs for reactivity with their baboon sera before our using them for this study. However, we consider it highly unlikely as the preimmune sera titers were uniformly low. Moreover, for this to explain the unex- Disclosures pectedly high humoral responses seen for the older baboons, they The authors have no financial conflict of interest. would all have to have had the same prior exposure and none of the younger animals would have been similarly affected. In other References words, these genetically heterogeneous baboons would have all 1. Gavazzi, G., and K.-H. Krause. 2002. Ageing and infection. Lancet Infect. Dis. been responding in the same way; this is highly improbable. Thus, 2: 659–666. 2. Ginaldi, L., M. F. Loreto, M. P. Corsi, M. Modesti, and M. De Martinis. 2001. it appears most likely that the response to LcrV is a primary one in Immunosenescence and infectious diseases. Microbes Infect. 3: 851–857. both age groups and that the higher titers of protective Abs seen in 3. Webster, R. G. 2000. Immunity to influenza in the elderly. Vaccine 18: 1686–1689. the older baboons is due to a difference in the manner in which 4. Hakim, F. T., F. A. Flomerfelt, M. Boyiadzis, and R. E. Gress. 2004. Aging, aging affects immune responsiveness in this NHP species. immunity, and cancer. Curr. Opin. Immunol. 16: 151–156. The Journal of Immunology 115

5. Oxman, M. N., M. J. Levin, J., G. R. Johnson, K. E. Schmader, S. E. Straus, 26. Polack, F. P., S. H. Lee, S. Permar, E. Manyara, H. G. Nousari, Y. Jeng, L. D. Gelb, R. D. Arbeit, M. S. Simberkoff, A. A. Gershon, L. E. Davis, et al. F. Mustafa, A. Valsamakis, R. J. Adams, H. L. Robinson, and D. E. Griffin. 2000. 2005. A vaccine to prevent herpes zoster and postherpetic neuralgia in older Successful DNA immunization against measles: neutralizing antibody against adults. N. Engl. J. Med. 352: 2271–2284. either the hemagglutinin or fusion glycoprotein protects rhesus macaques without 6. Vu, T., S. Farish, M. Jenkins, and H. Kelly. 2002. A meta-analysis of effective- evidence of atypical measles. Nat. Med. 6: 776–781. ness of influenza vaccine in persons aged 65 years and over living in the com- 27. Murthy, K. K., M. T. Salas, K. D. Carey, and J. L. Patterson. 2006. Baboon as a munity. Vaccine 20: 1831–1836. nonhuman primate model for vaccine studies. Vaccine 24: 4622–4624. 7. Hainz, U., B. Jenewein, E. Asch, K. P. Pfeiffer, P. Berger, and 28. Shearer, M. H., R. D. Dark, J. Chodosh, and R. C. Kennedy. 1999. Comparison B. Grubeck-Loebenstein. 2005. Insufficient protection for healthy elderly adults and characterization of immunoglobulin G subclasses among primate species. by tetanus and TBE vaccines. Vaccine 23: 3232–3235. Clin. Diag. Lab. Immunol. 6: 953–958. 8. Jefferson, T., D. Rivetti, A. Rivetti, M. Rudin, C. Di Pietrantonj, and 29. Attanasio, R., K. M. Brasky, S. H. Robbins, L. Jayashankar, R. J. Nash, and V. Demichelli. 2005. Efficacy and effectiveness of influenza vaccines in elderly T. M. Butler. 2001. Age related autoantibody production in a nonhuman primate people: a systematic review. Lancet 366: 1165–1174. model. Clin. Exp. Immunol. 123: 36–35. 9. de Bruijn, I. A., E. J. Remarque, W. E. Beyer, S. le Cessie, N. Masurel, and G. J. Ligthart. 1997. Annually repeated influenza vaccination improves humoral re- 30. Williamson, E. D., H. C. Flick-Smith, C. Lebutt, C. A. Rowland, S. M. Jones, sponses to several influenza virus strains in healthy elderly. Vaccine 15: E. L. Waters, R. J. Gwyther, J. Miller. P. J. Packer, and M. Irving. 2005. Human 1323–1329. immune response to a plague vaccine comprising recombinant F1 and V antigens. 10. McElhaney, J. E., G. S. Meneilly, K. E. Lechelt, B. L. Beattie, and Infect. Immun. 73: 3598–3608. R. C. Bleackley. 1993. Antibody response to whole-virus and split-virus influenza 31. Jones, S. M., F. Day, A. J. Stagg, and E. D. Williamson. 2000. Protection con- vaccines in successful aging. Vaccine. 11: 1055–1060. ferred by a fully recombinant sub-unit vaccine against Yersinia pestis in male and 11. Ben-Yedidia, T., L. Abel, R. Arnon, and A. Globerson. 1998. Efficacy of anti- female mice of four inbred strains. Vaccine 19: 358–366. influenza peptide vaccine in aged mice. Mech. Ageing Dev. 104: 11–23. 32. Jones, S. M., K. F. Griffin, I. Hodgson, and E. D. Williamson. 2003. Protective 12. Gahring, L. C., and W. O. Weigle. 1990. The effect of aging on the induction of efficacy of a fully recombinant plague vaccine in the guinea pig. Vaccine 21: humoral and cellular immunity and tolerance in two long-lived mouse strains. 3912–3918. Cell. Immunol. 128: 142–151. 33. Galimand, M., A. Guiyoule, G. Gerbaud, B. Rasoamanana, S. Chanteau, 13. Stacy, S., A. J. Infante, K. A. Wall, K. A. Krolick, and E. Kraig. 2003. Recall E. Carniel, and P. Courvalin. 1997. Multidrug resistance in Yersinia pestis me- Downloaded from immune memory: a new tool for generating late onset myasthenia gravis. Mech. diated by a transferable . N. Engl. J. Med. 337: 677–680. Ageing Dev. 124: 931–940. 34. Guiyoule, A., G. Gerbaud, C. Buchrieser, M. Galimand, L. Rahalison, 14. Frasca, D., R. L. Riley, and B. B. Blomberg. 2005. Humoral immune response S. Chanteau, P. Courvalin, and E. Carniel. 2001. Transferable plasmid-mediated and B-cell functions including immunoglobulin class switch are downregulated in resistance to streptomycin in a clinical isolate of Yersinia pestis. Emerg. Infect. aged mice and humans. Semin. Immunol. 17: 378–384. Dis. 7: 43–48. 15. Haynes, L., and S. M. Eaton. 2005. The effect of age on the cognate function of ϩ 35. Saikh, K. U., J. Sesno, P. Brandler, and R. G. Ulrich. 1998. Are DNA-based CD4 T cells. Immunol. Rev. 205: 220–228. vaccines useful for protection against secreted bacterial toxins? Tetanus toxin test

16. Miller, R. A., S. B. Berger, D. T. Burke, A. Galecki, G. G. Garcia, J. M. Harper, http://www.jimmunol.org/ case. Vaccine 16: 1029–1038. and A. A. Sadighi. 2005. T cells in aging mice: genetic, developmental, and biochemical analyses. Immunol. Rev. 205: 94–103. 36. Williamson, E. D., P. M. Vesey, K. J. Gillhespy, S. M. Eley, M. Green, and 17. Stacy, S., K. A. Krolick, A. J. Infante, and E. Kraig. 2002. Immunological mem- R. W. Titball. 1999. An IgG1 titre to the F1 and V antigens correlates with ory and late onset autoimmunity. Mech. Ageing Dev. 123: 975–985. protection against plague in the mouse model. Clin. Exp. Immunol. 116: 107–114. 18. Sambhara, S., A. Kurichh, R. Miranda, O. James, B. Underdown, M. Klein, 37. Eto, T., and H. Takahashi. 1999 Enhanced inhibition of hepatitis B virus pro- J. Tartaglia, and D. Burt. 2001. Severe impairment of primary but not memory duction by asialoglycoprotein receptor-directed interferon. Nat. Med. 5: 577–581. responses to influenza viral antigens in aged mice: costimulation in vivo partially 38. Romero-Steiner, S., D. M. Musher, M. S. Cetron, L. B. Pais, J. E. Groover, reverses impaired primary immune responses. Cell. Immunol. 210: 1–4. A. E. Fiore, B. D. Plikaytis, and G. M. Carlone. 1998. Reduction in functional 19. Harrod, T., M. Martin, and M. W. Russell. 2001. Long-term persistence and antibody avidity against Streptococcus pneumoniae in vaccinated elderly indi- recall of immune responses in aged mice after mucosal immunization. Oral Mi- viduals highly correlates with decreased IgG antibody avidity. Clin. Infect. Dis. crobiol. Immunol. 16: 170–177. 29: 281–8.

20. Kapasi, Z. F., K. Murali-Krishna, M. L. McRae, and R. Ahmed. 2002. Defective 39. Jayashankar, L., K. M. Brasky, J. A. Ward, and R. Attanasio. 2003. Lymphocyte by guest on September 27, 2021 generation but normal maintenance of memory T cells in old mice. Eur. J. Im- modulation in a baboon model of immunosenescence. Clin. Diag. Lab Immunol. munol. 32: 1567–1573. 10: 870–875. 21. Kennedy, R. C., M. H. Shearer, and W. Hildebrand. 1997. Nonhuman primate 40. Healy, D. L., J. Bacher, and G. D. Hodgen. 1983. A method of thymectomy in models to evaluate vaccine safety and immunogeneicity. Vaccine 15: 903–908. macaques monkeys. J. Med. Primatol. 12: 89–100. 22. MacPhail, M., J. H. Schickli, R. S. Tang, J. Kaur, C. Robinson, 41. Ershler, W. B., C. L. Coe, S. Gravenstein, K. T. Schultz, R. G. Klopp, M. Meyer, R. A. M. Fouchier, A. D. M. E. Osterhaus, R. R. Spaete, and A. A. Haller. 2004. and W. D. Houser. 1988. Aging and immunity in nonhuman primates: I. Effects Identification of small-animal and primate models for evaluation of vaccine can- of age and gender on cellular immune function in rhesus monkeys (Macaca didates for human metapneumovirus (hMPV) and implications for hMPV vaccine mulatta) J. Primatol. 15: 181–188. design. J. Gen. Virol. 85: 1655–1663. 23. Jeong, S.-H., M. Qiao, M. Nascimbeni, Z. Hu, B. Rehermann, K. Murthy, and 42. Jankovic, V., I. Messaoudi, and J. Nikolich-Zugich. 2003. Phenotypic and func- T.J. Liang. 2004. Immunization with hepatitis C virus-like particles induces hu- tional T cell aging in rhesus macaques differential behavior of CD4 and CD8 moral and cellular immune responses in nonhuman primates. J. Virol. 78: subsets. Blood 102: 3244–3251. 6995–7003. 43. Pitcher, C. J., S. I. Hagen, J. M. Walker, R. Lum, B. L. Mitchell, V. C. Maino, 24. Wolf, R. F., J. F. Papin, R. Hines-Boykin, M. Chavez-Suarez, G. L. White, M. K. Axthelm, and L. J. Picker. 2002. Development and homeostasis of T cell M. Sakalian, and D. P. Dittmer. 2006. Baboon model for West Nile virus infec- memory in rhesus macaque. J. Immunol. 168: 29–43. tion and vaccine evaluation. Virology 355: 44–51. 44. Lee, W.-W., K.-H. Nam, K. Terao, H. Akari, and Y. Yoshikawai. 2003. Age 25. Locher, C. P., S. A. Witt, B. G. Herndier, K. Tenner-Racz, P. Racz, and related increase of peripheral CD4ϩCD8ϩ double positive T lymphocytes in J. A. Levy. 2001. Baboons as an animal model for human immunodeficiency cynomolgus monkeys: longitudinal study in relation to thymic involution. Immu- virus pathogenesis and vaccine development. Immunol. Rev. 183: 127–140. nology 109: 217–225.