Attenuated poxvirus-based simian immunodeficiency virus (SIV) vaccines given in infancy partially protect infant and juvenile macaques against repeated oral challenge with virulent SIV

Abstract

An infant macaque model was developed to test pediatric vaccine candidates aimed at reducing HIV transmission through breast-feeding. Infant macaques were given multiple immunizations during the first 3 weeks of life with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins Gag, Pol, and Env (ALVAC-SIV or modified vaccinia virus Ankara [MVA]-SIV). After repeated daily oral inoculations with virulent SIVmac251 at 4 weeks of age, significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. Monkeys not infected after oral challenge in infancy were rechallenged at 16 months of age or older by repeated weekly oral SIV exposure; unimmunized animals were infected after fewer SIV exposures than were animals vaccinated with ALVAC-SIV or MVA-SIV. When infected, ALVAC-SIV- and MVA-SIV-vaccinated animals also had reduced viremia compared with unimmunized animals. The results of these investigations suggest that immunization of human infants with poxvirus-based HIV vaccine candidates may offer protection against early and late HIV infection through breastfeeding.

FIGURE 1.

Overview of infant vaccine groups and summary of outcomes. As described under Methods, newborn macaques were immunized within the first 3 weeks of life. MVA-SIV and ALVAC-SIV both express Gag, Pol, and Env of SIV, while ALVAC-vector was the empty vector. At 4 weeks of age, all animals were fed a total of 15 low doses of SIVmac251 (3 times per day for 5 consecutive days). P values (one-sided Fisher exact test) are for the comparison of immunized groups with the unvaccinated animals. Group C remained significantly different from the controls after adjusting for multiple comparisons using the Bonferroni correction. The relative risk of infection of ALVAC-SIV–immunized macaques was 0.43 (95% confidence interval, 0.22–0.82).

FIGURE 2.

SIVmac251 infection of infant macaques: effect of immunization on viremia and disease-free survival. All animals were challenged orally with SIVmac251 at 4 weeks of age (see Fig. 1). A, Plasma viral RNA levels for individual animals in each vaccine group. B, Average plasma viral RNA levels (calculated after log transformation) for only those animals that became persistently infected. Horizontal lines at plasma viral RNA levels of 7 and 8 log copies/mL (typical for unimmunized animals) are given for ease of comparison. C, Survival curves for the infants that became persistently infected. One ALVAC-SIV–immunized SIV-infected animal died early of what was considered an unrelated cause (aspiration pneumonia 2 weeks after SIV challenge).

FIGURE 3.

SIV-specific antibody responses and IFN-γ–producing cells in immunized infant macaques before and after SIVmac251 challenge. All animals were inoculated with SIVmac251 at 4 weeks of age (see Fig. 1). Levels of SIV-specific immunoglobulin G antibodies (A–C) were measured using a whole-virus ELISA (using 4-fold dilutions) and are presented as the reciprocal of the dilution that gave a signal above cutoff absorbance. A, SIV-specific antibody titers (with mean) at 4 weeks of age (ie, shortly before SIVmac251 challenge). B and C, Time course of the means (calculated after log transformation) of anti-SIV antibody titers; error bars on B represent SEM. C, In comparison with the unimmunized animals, the MVA-SIV–immunized SIVmac251-infected animals had the most rapid and most sustained antibody responses (P < 0.05 from 5 through 16 weeks of age; 2-way ANOVA with Bonferroni posttest), while the 6 ALVAC-SIV–immunized infected animals had a delay in antibody response that was still stronger than that of the unimmunized animals (P < 0.01 at 6 and 8 weeks of age). The CD8⁺ cell–depleted ALVAC-SIV–immunized animals had anti-SIV antibody titers indistinguishable from those of the undepleted ALVAC-SIV–immunized infected animals (P < 0.05 at all time points) but that were higher than those of the unimmunized animals (P < 0.05 at weeks 6 and 8). D–H, Because of the limited availability of PBMCs, samples from approximately one half of the animals in each group were used to quantitate SIV-specific IFN-γ–producing cells, while PBMCs from the remaining animals were used for quantitation of cytokine mRNA. SIV-specific IFN-γ–producing cells (expressed as SFC per 1 million PBMCs) were measured in PBMC samples from 25 infant macaques (8 unimmunized, 9 MVA-SIV immunized, and 8 ALVAC-SIV immunized) by an SIV Gag–specific ELISPOT assay, either before (D) or after (E) challenge. In 56 PBMC samples collected between 2 and 12 weeks after infection, SIV-specific IFN-γ–producing T cells were detected only in 6 samples from 4 MVA-SIV–immunized animals within the first 8 weeks after challenge; the positive values 2 weeks after challenge are indicated (E). F–H, PBMCs collected 2 weeks after oral SIVmac251 challenge (6 weeks of age) were available for quantitation of cytokine mRNA analysis using real-time PCR analysis. Values are expressed as increase compared with the average levels of cytokine mRNA in PBMCs from uninfected unimmunized infants at 4 weeks of age, using calculations described previously^,; the dashed line represents the cutoff value for increase (ie, average increase for the cytokine in PBMCs from the same uninfected unimmunized animals + 2 SDs). F–H, Relative mRNA levels for granzyme, perforin, and IFN-γ based on vaccine group and infection status (2, uninfected; +, infected). P values for comparison of values among infected animals were calculated using ANOVA with Tukey posttest comparison.

FIGURE 4.

Oral SIVmac251 challenge of juvenile macaques: SIV-negative survival rates and viremia. Unimmunized animals and animals previously immunized with SIV vaccines within the first 3 weeks of age (see Fig. 1) and now of juvenile age received weekly oral inoculations with diluted SIVmac251 (see Table 2). For the SIV-negative survival analysis (A), animals were considered as “infected” as soon as virus could be isolated from PBMCs, which coincided with detectable viral RNA levels and was each time the start of at least 4 consecutive weeks of detectable infectious virus and viral RNA. Kaplan–Meier analysis with log-rank test demonstrated that the immunized animals had lower infection rates than the 5 unimmunized animals (P = 0.005; hazard ratio of infection, 0.29 [95% confidence interval, 0.019–0.54]). MVA-SIV– and ALVAC-SlV–immunized animals had similar protection. B–D, Viral RNA levels per group, measured by bDNA (cutoff value, 125 viral RNA copies/mL). Horizontal lines at plasma viral RNA levels of 7 and 8 log copies/mL are given for ease of comparison. B, Animal 34004 was an unimmunized control that was not infected after the repeated exposures at 4 weeks of age (see Fig. 1); the other 4 animals had never been exposed to SIV. D, SIV-ALVAC–immunized animals 33859 and 33779 had been CD8⁺ cell depleted as indicated in Figure 1.