Reduced immunogenicity of a live Salmonella enterica serovar Typhimurium vaccine in aged mice

IntroductionNon-typhoidal Salmonella (NTS) is responsible for a high burden of foodborne infections and deaths worldwide. In the United States, NTS infections are the leading cause of hospitalizations and deaths due to foodborne illnesses, and older adults (≥65 years) are disproportionately affected by Salmonella infections. Due to this public health concern, we have developed a live attenuated vaccine, CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA), against Salmonella enterica serovar Typhimurium, a common serovar of NTS. Little is known about the effect of age on oral vaccine responses, and due to the decline in immune function with age, it is critical to evaluate vaccine candidates in older age groups during early product development.MethodsIn this study, adult (six-to-eight-week-old) and aged (18-month-old) C57BL/6 mice received two doses of CVD 1926 (109 CFU/dose) or PBS perorally, and animals were evaluated for antibody and cell-mediated immune responses. A separate set of mice were immunized and then pre-treated with streptomycin and challenged orally with 108 CFU of wild-type S. Typhimurium SL1344 at 4 weeks postimmunization.ResultsCompared to PBS-immunized mice, adult mice immunized with CVD 1926 had significantly lower S. Typhimurium counts in the spleen, liver, and small intestine upon challenge. In contrast, there were no differences in bacterial loads in the tissues of vaccinated versus PBS aged mice. Aged mice exhibited reduced Salmonella-specific antibody titers in the serum and feces following immunization with CVD 1926 compared to adult mice. In terms of T cell responses (T-CMI), immunized adult mice showed an increase in the frequency of IFN-γ- and IL-2-producing splenic CD4 T cells, IFN-γ- and TNF-α-producing Peyer’s Patch (PP)-derived CD4 T cells, and IFN-γ- and TNF-α-producing splenic CD8 T cells compared to adult mice administered PBS. In contrast, in aged mice, T-CMI responses were similar in vaccinated versus PBS mice. CVD 1926 elicited significantly more PP-derived multifunctional T cells in adult compared to aged mice.ConclusionThese data suggest that our candidate live attenuated S. Typhimurium vaccine, CVD 1926, may not be sufficiently protective or immunogenic in older humans and that mucosal responses to live-attenuated vaccines decrease with increasing age

Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium infections in sub-Saharan Africa

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30-43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63-74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa

Immunogenicity and efficacy following sequential parenterally-administered doses of <i>Salmonella</i> Enteritidis COPS:FliC glycoconjugates in infant and adult mice

<div><p>In sub-Saharan Africa, invasive nontyphoidal <i>Salmonella</i> (iNTS) infections with serovars <i>S</i>. Enteritidis, <i>S</i>. Typhimurium and I 4,[5],12:i:- are widespread in children < 5 years old. Development of an efficacious vaccine would provide an important public health tool to prevent iNTS disease in this population. Glycoconjugates of <i>S</i>. Enteritidis core and O-polysaccharide (COPS) coupled to the homologous serovar phase 1 flagellin protein (FliC) were previously shown to be immunogenic and protected adult mice against death following challenge with a virulent Malian <i>S</i>. Enteritidis blood isolate. This study extends these observations to immunization of mice in early life and also assesses protection with partial and full regimens. Anti-COPS and anti-FliC serum IgG titers were assessed in infant and adult mice after immunization with 1, 2 or 3 doses of <i>S</i>. Enteritidis COPS:FliC alone or co-formulated with aluminum hydroxide or monophosphoryl lipid A (MPL) adjuvants. <i>S</i>. Enteritidis COPS:FliC was immunogenic in both age groups, although the immune responses were quantitatively lower in infants. Kinetics of antibody production were similar for the native and adjuvanted formulations after three doses; conjugates formulated with MPL elicited significantly increased anti-COPS IgG titers in adult but not infant mice. Nevertheless, robust protection against <i>S</i>. Enteritidis challenge was seen for all three formulations when three doses were given either during infancy or as adults. We further found that significant protection could be achieved with two COPS:FliC doses, despite elicitation of modest serum anti-COPS IgG antibody titers. These findings guide potential immunization strategies that may be translated to develop a human pediatric iNTS vaccine for sub-Saharan Africa.</p></div

Anti-FliC IgG responses in adult and infant mice after immunization with <i>S</i>. Enteritidis COPS:FliC alone or formulated with different adjuvants.

<p>Infant and adult mice (<i>n</i> = 16–20/group) were immunized with PBS or <i>S</i>. Enteritidis COPS:FliC formulated alone (ɸ), adsorbed to alum, or admixed with MPL. (A) Serum anti-FliC IgG titers taken 12–14 days after each dose were determined by ELISA. Each point represents an individual mouse. Red squares indicate mice that succumbed to subsequent challenge. Bars represent the GMT for adults (grey) and infants (white), and were compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. ns, not significant. *<i>P</i> ≤ 0.05; **<i>P</i> ≤ 0.005; ***<i>P</i> ≤ 0.0005 for indicated comparisons. (B) Reverse cumulative distribution curves for post 3^rd immunization anti-FliC IgG titers for adults (grey circles) and infants (white circles) are depicted.</p

Isotype and avidity of anti-FliC IgG in adult and infant mice after immunization with <i>S</i>. Enteritidis COPS:FliC alone or formulated with different adjuvants.

<p>(A) The avidity of anti-FliC IgG in sera taken after each immunization (<i>n</i> = 8–10) was determined by sensitivity to urea treatment in an ELISA format. (B) Post 3^rd immunization FliC-specific serum IgG1 and IgG2b titers were determined by ELISA (<i>n</i> = 9–10). The ratio of the two titers is plotted. Each point represents an individual mouse. Bars represent the median for adults (grey) and infants (white) that was compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. P-values ≤ 0.05 were considered to be statistically significant; ns, not significant. *<i>P</i> ≤ 0.05; **<i>P</i> ≤ 0.005; ***<i>P</i> ≤ 0.0005 for indicated comparisons.</p

Survival for adult and infant mice immunized with 1, 2, or 3 doses of <i>S</i>. Enteritidis COPS:FliC adjuvanted with MPL and challenged with <i>S</i>. Enteritidis R11.

<p>Kaplan-Meier survival curves for adult-primed (A) or infant-primed (B) mice immunized with 1, 2, or 3 doses of PBS (black squares) or COPS:FliC (circles; grey and white color indicate adults and infants, respectively) after challenge with 1x10⁶ CFU/mL of <i>S</i>. Enteritidis R11 (<i>n</i> = 13–20/group). Survival curves were compared using log rank analysis. For comparisons between vaccinated mice and PBS controls, P-values ≤ 0.05 were considered to be statistically significant. ns, not significant; *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.0005.</p

Anti-COPS IgG responses in adult and infant mice after immunization with <i>S</i>. Enteritidis COPS:FliC alone or formulated with different adjuvants.

<p>(A) Infant and adult mice (<i>n</i> = 16–20/group) were immunized as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006522#pntd.0006522.g001" target="_blank">Fig 1</a>. Serum anti-COPS IgG titers taken 12–14 days after each dose were determined by ELISA. Each point represents an individual mouse. Red squares indicate mice that succumbed to subsequent challenge. Bars represent the GMT for adults (grey) and infants (white), and were compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. ns, not significant. *<i>P</i> ≤ 0.05; **<i>P</i> ≤ 0.005; ***<i>P</i> ≤ 0.0005 for indicated comparisons. (B) Reverse cumulative distribution curves for post 3^rd immunization anti-COPS IgG titers for adults (grey circles) and infants (white circles) are depicted. Dotted lines indicate the cut-off for seroconversion (50 EU/mL), which represents a 4-fold rise over the anti-COPS IgG GMT for PBS controls.</p

Isotype and avidity of anti-COPS IgG in adult mice after immunization with <i>S</i>. Enteritidis COPS:FliC formulated with MPL.

<p>(A) The avidity of anti-COPS IgG after the 3^rd immunization (<i>n</i> = 6) was determined by ELISA. (B) Post 3^rd immunization serum COPS-specific IgG1 and IgG2b titers were determined by ELISA. The ratio of the two titers is plotted. Each point represents an individual mouse. The bar represents the median.</p

Survival of adult and infant mice immunized with 3 doses of <i>S</i>. Enteritidis COPS:FliC alone or formulated with alum or MPL and challenged with <i>S</i>. Enteritidis R11.

<p>Kaplan-Meier survival curves for adult-primed or infant-primed mice immunized with 3 doses of either PBS (black squares), unadjuvanted COPS:FliC (white circles), alum-adsorbed COPS:FliC (grey triangles), or COPS:FliC admixed with MPL (black diamonds) after challenge with 1x10⁶ CFU/mL of <i>S</i>. Enteritidis R11 <i>(n</i> = 16–20/group). Survival curves were compared using log rank analysis. Adjustments for multiple comparisons were not made. For comparisons between vaccinated mice and PBS controls, P-values ≤ 0.05 were considered to be statistically significant. *<i>P</i> ≤ 0.0005.</p

Survival for adult mice immunized with 1 or 2 doses of <i>S</i>. Enteritidis COPS:FliC adjuvanted with MPL and challenged with <i>S</i>. Enteritidis R11 <i>∆fliC</i>.

<p>Kaplan-Meier survival curves for adult mice immunized with 1 or 2 doses of PBS (black squares) or COPS:FliC (grey circles) after challenge with 1x10⁶ CFU/mL of <i>S</i>. Enteritidis R11 <i>∆fliC</i> (<i>n</i> = 10–20/group). Survival curves were compared using log rank analysis. For comparisons between vaccinated mice and PBS controls, P-values ≤ 0.05 were considered to be statistically significant. ns, not significant; *<i>P</i> ≤ 0.005.</p