An age-old paradigm challenged: old baboons generate vigorous humoral immune responses to LcrV, a plague antigen

Abstract

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.

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 an SDS polyacrylamide gel and stained with Coomassie Blue. A duplicate gel was electrophoretically transferred and the resulting Western blot was developed using an antibody that recognizes the vector-encoded “his” tag.

FIGURE 2

Effects of age on the antibody 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 (Panel A) and mouse (Panel B) are shown. The titers from young animals are indicated using open bars (□) and those from old animals are indicated with the filled bars (■). 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 value ≤0.05 are indicated by an asterisk (*)).

FIGURE 3

Effects of aging on the antibody responses to a chimeric antigen, 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 antibodies and also for anti-TTFC antibodies using direct ELISAs. The end-point titers were determined and are presented for baboons (Panel A) and mice (Panel B). The titers from young animals are indicated using open bars ((□) and those from old animals are indicated with the filled bars ((■). Statistical significance of the differences between old and young within a treatment group was assessed as described in Materials and Methods.

FIGURE 4

Effects of aging on the generation of protective antibodies in baboons and mice. Panel 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 phosphate buffered saline (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 Panel A reports the number of animals surviving ten days for each of the time points tested. Panel B Young and old mice were immunized twice with Alum (negative control) or with one of the test immunogens, Y. pestis F1 antigen, LcrV, or a chimeric molecule LcrV::TTFC. They were then challenged (as described in Panel A) with 8000 cfu Y. pestis CO92. The percent survival (10 mice/group) was scored daily and is shown.

FIGURE 5

Effects of aging on the Ig subclass of antibodies produced in response to LcrV immunization of baboons. The primate sera from old (O) and young (Y) LcrV-immunized baboons were re-tested by ELISA using Ig subclass specific secondary antibodies 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 antibody used for IgG1 and IgG2. The levels of IgG3 and IgG4 reactive with LcrV were at baseline and are not shown.