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The Journal of Immunology, 2008, 181, 109 -115
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Seniors' Health

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

Sue Stacy*,{ddagger}, Amanda Pasquali*, Valerie L. Sexton{dagger}, Angelene M. Cantwell{dagger}, Ellen Kraig2,*,{ddagger} and Peter H. Dube{dagger},{ddagger}

* Department of Cellular and Structural Biology, {dagger} Department of Microbiology and Immunology, and {ddagger} Barshop Center for Longevity Studies, University of Texas Health Science Center, San Antonio, TX 78229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune senescence in the elderly results in decreased immunity with a concomitant increase in susceptibility to infection 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 animals has not been established. Toward that end, immunity to LcrV, a protective Ag from Yersinia pestis, 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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Elderly individuals show diminished immune responses, making them significantly more susceptible to infections and cancer (reviewed in Ref. 1, 2, 3, 4). In addition, vaccination protocols are typically less efficacious in the elderly and although higher doses of immunogen may enhance the response, it is typically still lower than the one elicited in younger individuals (5, 6, 7, 8). Deficits in the ability of older subjects to generate immune responses, particularly to "new" Ags that they have not previously encountered, have been widely reported. In contrast, immune memory to Ags encountered in one’s youth does survive aging and can be recalled in old age (9, 10). Given the current demographic composition in the U.S., the numbers of aging individuals will continue to grow and they already comprise a significant "at risk" population. Thus, it is critically important to develop and test protocols for enhancing immunity particularly to "new" Ags in old, as well as in young, individuals.

Most of the research into the effects of aging on immunity has incorporated rodent models and, for the most part, analogous age-associated deficiencies of cellular and humoral immunity have been seen (11, 12, 13, 14, 15, 16). For example, the ability to generate an immune response to a "new" Ag or epitope not previously encountered is significantly diminished in older animals. In contrast, memory immunity to Ags encountered in one’s youth appears largely intact (17, 18, 19, 20). Similarly, the age-associated loss of immunity can be overcome by giving multiple immunizations or higher doses of the Ag. This further substantiates the imperative to test vaccine protocols for their effectiveness in both old and young subjects.

Although many vaccines are first tested in rodents, this may not be ideal for protection studies because mice are resistant to many human pathogens (like HIV) due to sequence differences in their cellular receptors. Thus, many vaccine protocols for use in humans have been tested in nonhuman primate (NHP)3 models (21, 22, 23, 24, 25, 26). However, the vast majority of these studies have been undertaken in young or middle-aged NHPs and none of these primate models has been validated for use in testing vaccines for efficacy in older individuals. Thus, in the current study, the ability of young and old NHPs to respond to a "new" Ag has been assessed. We chose to focus on the baboon, Papio hamadryas, for several reasons. It is an excellent primate model system due both to its close genetic relatedness ({approx}96% DNA homology) and the similarity of its immune system to humans (27). For example, unlike macaques and some other monkeys, baboons resemble humans and chimpanzees in exhibiting four IgG subclasses (28). Moreover, because baboons breed well in captivity, they are more readily available than some other NHPs. Baboons are being used extensively in infectious disease 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 to humans (29), but there are no studies that assess the effects of aging on humoral immunity. Fortunately for this study, the Southwest National Primate Research Center in San Antonio maintains the largest colony of baboons worldwide; it consists of >3700 individual animals, including a geriatric cohort.

Given that aging most dramatically affects immune responses to Ags not previously encountered by the subject, it was imperative to select an immunogen that would elicit a primary response in the baboon colony. Thus, we chose LcrV, a protein Ag from Yersinia pestis, the causative agent of bubonic plague. Y. pestis is the most virulent bacterial pathogen currently known and in geographic areas where it is endemic in rodent populations, including the southwestern 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 vaccine for use in the U.S., a new subunit vaccine, which includes LcrV as one of its components, is showing promise (30, 31, 32). By incorporating LcrV in this study, we could assess both the titer of reactive Ab produced and its ability to protect against infection. This was considered a significant advantage as there is growing concern that plague may re-emerge as a significant danger to human health due to the recent identification of multidrug resistant strains of Y. pestis, thus, making the development of an effective vaccine a priority (33, 34). Yet, in no case has a protein-based vaccine for plague been tested in older animals whose immune responses are likely compromised. Thus, these studies will provide the first data on the effects of aging on the humoral immune response to LcrV in two different species, mice and baboons. Moreover, although the Y. pestis LcrV Ag was chosen for this study, our findings should be generalizable to all protein-based and subunit vaccines and should also provide important insights into the use of NHP models for testing vaccine efficacy in the elderly.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Antigens

To produce recombinant immunogens, wild-type CO92 genomic DNA, containing the pMT and pCD1 virulence plasmids, was used as the template in PCR to amplify the caf1 (F1) and lcrV genes. The primers used for F1 were: 5'-GAC GAC GAC GAC AAG GAT TTA ACT GCA AGC (forward) and 5'-TCA CTA TCA TCA TTA TTG GTT AGA TAC GGT TAC (reverse). The primers designed to amplify the LcrV coding sequence were: 5'-GAC GAC GAC GAC AAG ATG ATT AGA GCC TAC GAA CAA AAC CCA CAA CAT (forward) and 5'-AAG ACC TTG TGA GCA TCC TCG (reverse). In addition, each of the forward primers included sequences added at the 5' end to introduce an enterokinase cleavage site; this is shown in italics in the primer sequences above. The LcrV and F1 PCR products were TA cloned into the bacterial expression vector pQE-30UA vector (Qiagen) which provides an amino terminal histidine tag to facilitate protein purification. The chimeric Ag, LcrV::TTFC was created by subcloning the tetanus toxoid fragment C (TTFC) coding sequence in-frame downstream from LcrV at the BamHI site in pQE-30; the original TTFC clone was provided by Dr. Robert Ulrich (Army Medical Research Institute of Infectious Diseases, Frederick, MD) and is described elsewhere (35). All three inserts were sequenced by the University of Texas Health Science Center at San Antonio DNA Core Facility to ensure that the proper sequence had been cloned as a functional translational fusion.

The recombinant clones were transformed into Escherichia coli BL21 (DE3) pLys S and expression was induced by the addition of isopropyl β-D-thiogalactoside. Cell pellets were collected and disrupted by sonication and the lysate was cleared by centrifugation at 20,000 x G for 20 min. Recombinant proteins were purified from the soluble fraction using affinity chromatography on nickel chelating resin (Pharmacia) and eluted with 500 mM imidazole. The fractions containing recombinant protein were further purified by gel filtration followed by ion exchange chromatography on a resource Q column before being eluted with 500 mM NaCl. Positive fractions were pooled and the buffer was exchanged to a PBS solution (pH 7.4) on a gel filtration column. Contaminating endotoxin was removed with polymixin agarose (Sigma-Aldrich) and the protein was stored in aliquots at –80°C before use in immunization protocols.

To demonstrate purity of the recombinant proteins, a sample of each was fractionated by size on an SDS-polyacrylamide gel and stained (see Fig. 1). In addition, a duplicate gel was electrophoretically transferred to Duralon Membrane (Millipore) and the resulting Western blot was incubated with an Ab against the vector-encoded "his" tag (Qiagen) and developed using an alkaline phosphatase conjugated secondary Ab.


Figure 1
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  		FIGURE 1. Preparation of protein immunogens. The recombinant proteins were prepared in a prokaryotic expression system and purified, as described in Materials and Methods. In order to demonstrate purity, they were fractionated on a SDS polyacrylamide gel and stained with Coomassie Blue. A duplicate gel was electrophoretically transferred and the resulting Western blot was developed using an Ab that recognizes the vector-encoded "his" tag.

 
Baboon immunization

The purified recombinant proteins were absorbed to 25% alum (Vol/Vol) and used to immunize young (21/2 years) and old (19–24 years) baboons from the pedigreed colony at the Southwest National Primate Research Center. Each animal was vaccinated by i.m. injection with 100 µg of LcrV or the chimeric Ag, LcrV::TTFC. Sera were obtained from each animal before immunization (preimmune) and at intervals following the primary immunization (4 and 8 wk). All of the baboons received a second immunization with 100 µg of recombinant LcrV in Alum 6 mo subsequent to the primary exposure. The animals were bled 2 and 6 wk after the secondary inoculation.

Mouse immunizations

C57BL/6 mice were obtained from the NIA contract colony (Harlan Sprague Dawley) and were used at either 3 mo of age (young) or 19–21 mo (old). The old and young mice were immunized with 10 µg of LcrV, LcrV::TTFC, or F1 Ag in alum. The mice were bled from the retro-orbital sinus before immunization and at intervals post primary (4 and 9 wk) and secondary (2.4 and 5.4 wk) exposures. The sera were frozen at –20°C for subsequent analyses.

Measuring Ab titers

Direct ELISAs were developed to measure the levels of serum Abs specific for LcrV and F1 using the recombinant proteins generated above. For assessing reactivity to TTFC, a recombinant protein lacking the "his" tag was obtained from commercial sources (Roche). In all cases, Immunosorb 96-well plates (Nunc) were coated with the appropriate Ag (5 µg/ml for LcrV and F1, 10 µg/ml for TTFC). A dilution series of each baboon or murine sera was prepared and incubated with the Ag. Specific Ab binding was detected with HRP-conjugated anti-monkey IgG (Kirkegaard & Perry Laboratories) or rabbit anti-mouse IgG (Sigma-Aldrich). The ELISAs were developed by the addition of a chromogenic substrate ABTS and the absorbance at 410 nm was determined. Titers were fitted to a sigmoidal curve (GraphPad Prism 5) and the end-point titers at 0.1 OD above background were determined by interpolation.

Statistically significant differences between young and old animals were determined by one-way ANOVA (p ≤ 0.05). To satisfy the assumption of variance equivalence among treatment groups, a log base ten transformation of the data was performed. The statistical significance of differences in responses between the young and old animals in a given treatment group was then assessed by one-way ANOVA on the transformed data.

ELISAs were also performed using HRP-conjugated sheep Abs specific for human IgG1 (AP006), IgG2 (AP007), IgG3 (AP008), and IgG4 (AP009) from The Binding Site. For this purpose, ELISA plates were coated with recombinant LcrV as described above and then incubated with baboon and mouse sera diluted 1/500; this dilution was within the linear range for total IgG. The secondary Abs were then tested in duplicate wells at three different dilutions (1/1000, 1/3000, and 1/9000). The plates were developed and read as described above.

Measuring the protective capacity of the baboon Ab response

To assess the protective capacity of the baboon sera, pools of individual sera from a given time point were tested for the ability to protect mice from Y. pestis challenge; this is the US Public Health Service approved method for measuring protective immune responses to this virulent pathogen (30). In brief, 0.5 ml of each pool (from a given time point) was used to passively immunize female CD-1 mice (5/group). 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 sedated with avertin 0.5 mg/kg i.p. and challenged intradermally with 119 cfu of CO92 in the ear as described (36). The bacteria were originally obtained from the Centers for Disease Control and Prevention Select Agent Distribution Activity and were grown overnight 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 challenged intradermally with 8000 cfu Y. pestis CO92. The percent survival (10 mice/group) was scored daily.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
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 in a NHP model, the Y. pestis LcrV gene was cloned into a prokaryotic expression vector and protein immunogen was prepared. To demonstrate purity of the recombinant LcrV, it was subjected to SDS-PAGE and Western blot analysis using an Ab against the vector-encoded "his" tag. As shown in Fig. 1, there was one major protein species detected and it was of the size predicted for the "his"-tagged LcrV. The purified LcrV was then absorbed to alum, an adjuvant approved for use in humans, and used to immunize young (21/2 years) and old (19–24 years) baboons from the pedigreed colony at the Southwest National Primate Research Center. Sera were obtained from each baboon before immunization (preimmune) and at 4 and 8 wk following the primary inoculation. The titer of LcrV reactive Abs in each sample was determined by direct ELISA and the end-point titers were calculated using the GraphPad Prism 5 program; the results are shown in Fig. 2. Because end-point titers could not be determined for the preimmune sera, all individual baboon samples were retested at one dilution point within the linear range (1/1200) to allow direct comparison across samples; these data are shown in Table I. As expected, the preimmune titers to LcrV were relatively low, although one of the young baboons did have a slightly elevated level (Table I). Upon immunization, the titers increased in all animals relative to the preimmune levels, but, unexpectedly, the elderly baboons responded at least as well as the younger animals; there was no age-associated loss in immune competence (Fig. 2A).


Figure 2
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  		FIGURE 2. Effects of age on the Ab response to LcrV in baboons and mice. Young and old animals were immunized twice with Y. pestis LcrV in alum and were bled at the indicated time points after the primary and secondary immunizations. ELISAs were performed in duplicate with diluted sera on plates coated with recombinant LcrV and end-point titers were determined using the GraphPad Prism 5 program. The data for both baboons (A) and mouse (B) are shown. The titers from young animals are indicated using open bars ({square}) and those from old animals are indicated with the filled bars ({blacksquare}). Statistically significant differences between young and old animals within a treatment group were determined by one-way ANOVA as described in Materials and Methods (p ≤ 0.05 are indicated by an asterisk (*)).

 

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  		Table I. Ab responses of individual baboons immunized with LcrV

 
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 possibility, 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.

Response to a chimeric Ag confirms lack of immunosenescence in baboons

To pursue this question further, old and young animals were immunized with a second immunogen, a chimeric molecule composed of LcrV and TTFC; the purity of this immunogen was similarly demonstrated (Fig. 1). Because they were immunized with a chimeric molecule, the baboons and mice should produce Abs to each of the individual components, LcrV and TTFC. Therefore, ELISAs were performed independently with these two Ags (Fig. 3). In both cases, old baboons responded as well, if not better, than the young animals. In mice, as expected, the pattern was reversed; young responded better than old.


Figure 3
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  		FIGURE 3. Effects of aging on the Ab responses to a chimeric Ag, LcrV::TTFC. Old and young animals were immunized with recombinant chimeric LcrV::TTFC protein in alum. Sera, collected before immunization and at the indicated times thereafter, were tested for the presence of anti-LcrV Abs and also for anti-TTFC Abs using direct ELISAs. The end-point titers were determined and are presented for baboons (A) and mice (B). The titers from young animals are indicated using open bars ({square}) and those from old animals are indicated with the filled bars ({blacksquare}). Statistical significance of the differences between old and young within a treatment group was assessed as described in Materials and Methods.

 
Effects of age on secondary responses in baboons and mice

In mice and humans, boosting of the immune response is expected 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 had been generated. Briefly, the mice and baboons previously immunized with LcrV were given a secondary immunization and the titers of the resulting responses were measured by ELISA. The secondary responses were higher for both young and old mice, as expected (Fig. 2). In baboons, the secondary responses were boosted for the young animals, but were not enhanced over the primary responses for the older animals (Fig. 2 and Table I). Thus, in both cases, memory immune responses were generated but again, the baboon profile does not look like the one typically seen in mice or humans.

Elicitation of protective humoral responses in old and young baboons

Even though the elderly baboons showed higher Ab responses to LcrV immunization when compared with the young, it was possible that the Abs produced by the aged animals would not be as capable of neutralizing virulent bacteria. To test this possibility, mice were passively immunized with baboon sera pooled from a given time point and were then challenged with virulent Y. pestis. The mice were scored daily; a baboon serum was considered protective if at least 4/5 recipient mice (≥80%) survived 10 days. Protection was not seen in the mice receiving young preimmune sera (0% survival, Fig. 4A), old preimmune sera (20% survival, Fig. 4A), or PBS (0% survival, data not shown). Thus, the infectious dose delivered was sufficient to cause terminal disease and the baboons did not have significant pre-existing immunity to Y. pestis.


Figure 4
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  		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.

 
The kinetics of survival (protection) is shown for the serum samples taken 4 wk after the primary immunization (second panel in Fig. 4A). As seen with the preimmune samples, the young immune sera provide little protection. However, transfer of the sera from old baboons, even after a single immunization, provided high level protection from Y. pestis challenge. The survival data for all time points taken after primary immunization are summarized (table on right, Fig. 4A). The protective capability of the old baboon sera had diminished by 8 wk after immunization. This was somewhat surprising because there were still relatively high titers at this time. However, the protection assay is more physiologically relevant; it measures the functional capability of the Abs elicited.

The ELISA titers were also generally predictive of protection after secondary exposure. For both young and old baboons, relatively high titers were seen within 2 wk after immunization and these Abs were protective (third panel of Fig. 4A). However, as seen with the primary immunization, protection declined relatively quickly and was below the 80% cut-off by 6 wk postsecondary immunization (Fig. 4A, summary table). Thus, these data confirm that the old baboons generate Abs to LcrV that are both high titer and protective in vivo.

Elicitation of protective humoral responses in old and young mice

For comparison, the generation of protective responses in mice was tested directly by immunization and subsequent challenge with virulent Y. pestis, following the secondary exposure. As shown in Fig. 4B, protection correlated with Ab titer, for the time points tested. As expected, old mice were much more susceptible to challenge, presumably due to the lower titers of anti-LcrV Abs elicited. This finding was generalizable to three different immunogens, LcrV, LcrV::TTFC, and another plague vaccine component, F1. Thus, as expected, aging negatively impacts immunity in mice. In contrast, older baboons appeared to have a healthier immune system than the younger animals.

Isotype profiles of LcrV-specific Abs produced by young and old baboons

There have been well documented changes in cytokine 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 generate Abs to LcrV that are high titer, protective in vivo, and of similar Ig subclasses to those seen in younger animals.


Figure 5
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  		FIGURE 5. Effects of aging on the Ig subclass of Abs produced in response to LcrV immunization of baboons. The primate sera from old (O) and young (Y) LcrV-immunized baboons were retested by ELISA using Ig subclass-specific secondary Abs from The Binding Site. For this experiment, the baboon sera were all used at a single dilution point (1/500) previously shown to be within the linear range of the assay. The average 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 LcrV were at baseline and are not shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It has been generally accepted that all animals undergo immunosenescence associated with a decline in the ability of older individuals to mount a protective immune response to Ags not previously encountered. This report demonstrating that older baboons are at least as good as young animals at generating a protective humoral response to LcrV challenges that age-old paradigm. We considered several factors that might have contributed to this surprising finding. First, it was possible, although highly unlikely, that the specific baboon ages chosen for the analysis were not appropriate. The young baboons were 21/2–3 years old when first immunized, which approximates 71/2–9 year old humans. As juveniles, they would have attained immunological maturity, but not sexual maturity. This age group was chosen based on extensive baboon data demonstrating that they would be immunologically competent (much like school-age children). For example, Attanasio and colleagues (29) demonstrated that the levels of serum Ig are close to adult levels by 1 year of age in baboons. In addition, in a West Nile virus vaccine study, a 3-year-old juvenile baboon was immunized and generated IgG levels as high or higher than those seen in young adults (5.5–9 years of age) (24). In fact, even fetal baboons are capable of responding with IgG responses upon immunization with hepatitis B virus Ags (37). The older animals (19–24 years of age) were chosen to be ≥2/3 of the average live span, which would be roughly equivalent to 57- to 72-year-old humans and to 24-mo-old mice. If immunosenescence occurred equivalently in baboons, these animals should have shown a significant decrease in their Ab titers upon immunization, relative to the younger animals. Instead, they exhibited just the opposite; old baboons responded at very high levels to immunization.

In mice, the decline in immune capability has been shown to be a gradual one; middle-aged animals produce a humoral response that is intermediate, falling between the very low levels seen in older mice and the much higher levels produced by younger ones (14, 17). Given that the old baboons used in this study were not extremely old or geriatric, we expected detectable induced titers, but these should, if the paradigm were correct, have been lower than those achieved in younger animals. Clearly, this was not the case; aging appears to have less of an effect on humoral immunity in baboons than in other species examined to date.

We next explored the possibility that the old baboons had been previously exposed to LcrV. If so, the immunization for this study would have boosted an existing memory response, which would potentially account for the unexpectedly high responses in the elderly baboons. However, we consider it highly unlikely for several reasons. First, we chose the Ag, LcrV, specifically because the animals should have been naive. Y. pestis has not been reported at the Southwest National Primate Research Center and had any animal been infected, it would likely have died previously. Moreover, due to the extreme susceptibility of NHP to Yersinia spp, it is also unlikely that these animals were exposed to the related enteropathogenic species of Yersinia. Second, none of our animals had been previously enrolled in a study involving any Ag from this bacterium. Third, another Ag, TTFC, as part of the chimeric molecule, also elicited high responses from the older baboons. We cannot unconditionally eliminate the possibility that the old animals had been exposed to some other bacterium or Ag that elicited a response that would be cross-reactive with LcrV and/or TTFC. However, we consider it highly unlikely as the preimmune sera titers were uniformly low. Moreover, for this to explain the unexpectedly high humoral responses seen for the older baboons, they would all have to have had the same prior exposure and none of the younger animals would have been similarly affected. In other words, these genetically heterogeneous baboons would have all been responding in the same way; this is highly improbable. Thus, it appears most likely that the response to LcrV is a primary one in both age groups and that the higher titers of protective Abs seen in the older baboons is due to a difference in the manner in which aging affects immune responsiveness in this NHP species.

In humans and rodents, immunosenescence is accompanied by decreased Ab affinity, due to diminished somatic mutation in older individuals (38). A decrease in affinity for Ag would be expected to negatively impact its ability to function optimally in response to a pathogen and may well compromise its neutralizing capabilities. Thus, we might have expected that the anti-sera from old baboons, despite their relatively high titer Ab content, would have proven less efficacious in the passive transfer protection assay. However, this was not the case; the sera from old baboons, even after a single immunization, were significantly more potent in protecting from Y. pestis challenge than were the sera from younger individuals.

Other aspects of the aging immune system in baboon have been studied, with mixed results. For example, old baboons show a decrease in the number of B cells and an increase in T cell numbers with age (39); this is unlike humans. However, some aspects of the immune dysregulation seen in humans and rodents may also occur in baboons because the levels of serum autoantibodies do increase with age (29), suggesting that tolerance mechanisms may be impacted by aging. By comparing the aging human immune system, which shows profound deficiencies in immunity, to the baboon immune system, which is less impacted, we may gain important insights into immunosenescence and novel mechanisms to slow the process down. Importantly, given that the expected decrease in immunity to newly encountered Ags does not occur in aging baboons, other NHP models should be considered for testing vaccine compositions and immune-based therapeutics, particularly for their use in the elderly. There have been only a few studies in NHP to assess the effects of aging on the immune system. Thus far, the results have been quite mixed, but there are parallels seen to humans in several of the NHP species tested. For example, thymus involution does occur with aging in macaques (40). Moreover, lymphocyte proliferative responses and humoral immunity have been shown to decline with age in these monkeys, but the changes did not entirely mimic humans (41). Additional studies in rhesus macaques have shown that CD28 expression declines with age as it does in humans but signaling and cell cycle regulation appear to differ (42, 43). Similarly, in aging cynomolgus monkeys, an increase in double-positive (CD4+CD8+) T cells was demonstrated (44), suggesting a possible loss of thymic selection. Before selection of a particular NHP for use in testing vaccines or therapeutics, the responses to standard immunization protocols should be tested in young and old individuals to assess whether they show immunosenescence, like humans, or fail to show age-associated losses in humoral immunity, like baboons.


   Acknowledgments
 
We express our gratitude to Dr. Karen Rice and Sabrina Chatman for providing expertise and for coordinating the nonhuman primate protocols and procedures. We also thank Dr. William Morgan for very helpful advice in the statistical analysis of these data. In addition, we are grateful to Dr. Robert Ulrich for providing the TTFC clone. Lastly, we express our appreciation to Dr. Philip LoVerde whose laboratory screened the Binding Site secondary Abs for reactivity with their baboon sera before our using them for this study.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funding from the Southwest National Primate Center (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. Back

2 Address correspondence and reprint requests to Dr. Ellen Kraig, Cellular and Structural Biology MC 7762, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: kraig{at}uthscsa.edu Back

3 Abbreviations used in this paper: NHP, Nonhuman primate; TTFC, Tetanus toxoid fragment C. Back

Received for publication July 27, 2007. Accepted for publication April 22, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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