JAIDS Journal of Acquired Immune Deficiency Syndromes:
Attenuated Poxvirus-Based Simian Immunodeficiency Virus (SIV) Vaccines Given in Infancy Partially Protect Infant and Juvenile Macaques Against Repeated Oral Challenge With Virulent SIV
Van Rompay, Koen K. A DVM, PhD*; Abel, Kristina PhD*†; Lawson, Jonathan R BS*; Singh, Raman P BS*; Schmidt, Kimberli A AA*; Evans, Thomas MD‡; Earl, Patricia PhD∥; Harvey, Danielle PhD§; Franchini, Genoveffa MD¶; Tartaglia, James PhD#; Montefiori, David PhD**; Hattangadi, Shilpa MD∥; Moss, Bernard MD, PhD∥; Marthas, Marta L PhD*
From the *California National Primate Research Center, University of California at Davis, Davis, CA; †Center for Comparative Medicine, University of California at Davis, Davis, CA; ‡Division of Infectious Diseases, School of Medicine, University of California at Davis, Davis, CA; §Division of Biostatistics, Department of Epidemiology and Preventive Medicine, School of Medicine, University of California, Davis, CA; ∥Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD; ¶Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD; #Aventis Pasteur, Ltd., Toronto, Ontario, Canada; and **Duke University Medical Center, Durham, NC.
Received for publication June 11,2004; accepted August 12,2004.
Supported by Public Health Science grant RR00169 from the National Center for Research Resources and NIH/NIAID grants AI39109 and AI46320 (to M.L.M.); M.L.M. is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation.
Reprints: Marta L. Marthas, California National Primate Research Center, University of California at Davis, Davis, CA 95616 (e-mail: email@example.com).
Summary: 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.
Despite the progress in reducing intrapartum transmission using short-term antiretroviral regimens, breast-feeding in developing countries continues to be a considerable risk factor for postnatal mother-to-child transmission of HIV.1-3 Although prolonged administration of antiviral drugs to nursing infants has the potential to reduce HIV transmission,4 their cost, risk of toxicity, emergence of resistance, and need for regular administration are limiting factors. Thus, there is an urgent need for a vaccine that, when administered to the infant shortly after birth, could protect against HIV transmission via breast-feeding (see review by Safrit et al5).
Simian immunodeficiency virus (SIV) infection in infant macaques is a highly relevant animal model of pediatric HIV infection because of its many similarities in disease pathogenesis, immunology, and physiology (reviewed by Van Rompay and Marthas6 and Van Rompay et al7). This pediatric animal model has also been used to test novel drug strategies and pediatric HIV vaccine candidates.8-10 Previously, we showed in this animal model that a poxvirus-based SIV vaccine was immunogenic but did not protect against a relatively high-dose SIV challenge.10 However, HIV transmission via breast-feeding occurs through multiple exposures of infants to lower doses of virus. Thus, in the current study, we evaluated the relative efficacy of poxvirus-based vaccine candidates against repeated low-dose exposures with the highly virulent SIVmac251. We demonstrate that protection against repeated low-dose oral exposure to virulent SIV can be achieved in infant and juvenile macaques by prior immunization with modified vaccinia virus Ankara (MVA)- and ALVAC-based SIV recombinant viruses.
Animals and Immunization Regimens
Newborn rhesus macaques (Macaca mulatta) negative for HIV type 2, SIV, type D retrovirus, and simian T-cell lym-photropic virus type 1 were hand-reared in a primate nursery at the California National Primate Research Center. Animals were housed in accordance with American Association for Accreditation of Laboratory Animal Care standards. We adhered to the “Guide for Care and Use of Laboratory Animals.”11 When necessary, animals were immobilized with 10 mg/kg ketamine hydrochloride (Parke-Davis, Morris Plains, NJ) injected intramuscularly. Genetic assessment of the major histocompatibility complex class I alleles MamuA*01 and MamuB*01 was performed using a PCR-based technique.12,13 In the present study, because animals were randomly assigned at birth, the frequency of the MamuA*01 and MamuB*01 alleles was similar to that among our rhesus macaque colony (∼25%). There was no association between the presence of the major histocompatibility complex class I MamuA*01 and MamuB*01 alleles and infection status or viremia after oral challenge (data not shown).
During these experiments, 1 of several SIV vaccines was administered to infant macaques as summarized in Figure 1. Group A animals were unimmunized controls. Group B animals received MVA expressing SIVmac239 Gag, Pol, and Env (MVA-SIV) either at weeks 0 and 3 (1 vector [MVA/SIVgpe]; 9 animals) or at weeks 0, 2, and 3 (2 vectors [MVA/SIV239gagpol and MVA/SIV239env]; 8 animals). Because there was no statistically significant difference in outcome (7 of 9 and 4 of 8 animals became infected, respectively; Fisher exact test, P = 0.33), the results for these MVA-SIV-immunized animals were pooled. Each immunization consisted of 1 mL administered intramuscularly (250 μL in each of 4 limbs; 1 × 108 infectious units [IU] of each recombinant construct/mL). The construction of MVA/SIVgpe10 and MVA/SIV239gagpol14 has been described previously. For the construction of MVA/SIV239env, the plasmid pSP72-239-3′ (gift from R. Desrosiers) was the source of the SIVmac239 env gene. The gene was truncated at the C-terminus to delete 146 amino acids and cloned into the MVA expression plasmid pLW17,15 a transfer vector that directs insertion into deletion II of MVA under control of the mH5 promoter. All recombinant MVAs were constructed using standard techniques, including multiple rounds of plaque purification.15 Viruses were amplified in chicken embryo fibroblast cells and purified through a sucrose cushion. All viral stocks were determined to be free of nonrecombinant MVA by PCR amplification of the region of the genome containing the inserted SIV gene. Efficient expression and processing of Env and Gag proteins was demonstrated by metabolic labeling of infected BSC-1 cells, a monkey fibroblast cell line, followed by immunoprecipitation using serum from an SIV-infected monkey (data not shown). Expression of the gag and env genes in both MVA/SIVgpe and MVA/SIV239gagpol was completely stable. However, the stock of MVA/SIV239env used in this study contained ∼20% of non-Env-expressing virus particles, as determined by immunostaining of >800 individual infectious particles, despite the fact that the stock contained no nonrecombinant MVA. The mechanism of loss of Env expression in a subpopulation of this virus stock is not known. The practical result of this is a reduction of the effective dose of MVA/SIV239env to 0.8 × 108 IU. Group C animals and group D animals received 1 mL (1 × 108 IU/mL) of canarypox vector expressing SIVmacl42 Gag, Pol, and Env (ALVAC-SIV)16 administered intramuscularly (250 μL per limb) at 0, 2, and 3 weeks of age. Group E animals received parental CP pp ALVAC-vector16 administered at the same dose as ALVAC-SIV.
To monitor immunocompetence, all infants were immunized subcutaneously with cholera toxin B subunit at 6 and 16 weeks of age and intramuscularly with diphtheria and tetanus toxoids as described previously.17
Oral SIVmac251 Challenge
The uncloned SIVmac251 stock (with internal reference number 5/98), propagated on rhesus peripheral blood mono-nuclear cells (PBMCs), had a titer of 105 50% tissue culture infective doses (TCID50) and 1.4 × 109 SIV RNA copies/mL. This stock has many envelope variants, as determined by heteroduplex mobility assay (unpublished data). As described previously,18 we developed a repeated low-dose exposure model in which at 4 weeks of age, infant macaques are handheld and bottle-fed SIVmac251, diluted in a 1:1 mixture of RPMI-1640 medium and isotonic sucrose, a total of 15 times (3 times per day for 5 consecutive days). The sucrose in this mixture did not affect viral infectivity titers.18 On the basis of the results of a titration study (Table 1), each infant inoculation consisted of 2 mL of a 1:20 dilution of the SIVmac251 stock (∼104 TCID50 and 1.4 × 108 viral RNA copies per dose).
Juvenile macaques were rechallenged orally in 1 of 2 ways: “high-dose” challenge, animals received 2 oral inoculations (24 hours apart) with 1 mL of undiluted SIVmac251-5/98 stock (105 TCID50 per dose) during ketamine anesthesia; and “repeated low-dose” challenge, juvenile macaques received oral inoculations via a needleless syringe with 2 mL of SIVmac251 (of a stock with lot number 2/02, grown similarly to SIVmac251-5/98, and with a titer of 105 TCID50 and 0.9 × 109 SIV RNA copies/mL) diluted 1:100 in RPMI-1640 medium and sucrose (ie, 2000 TCID50 per dose) twice daily every Friday (8 AM and 2 PM) without chemical anesthesia. This experiment included 4 naive juvenile macaques (ie, never exposed to SIV). Virus inoculations in individual animals were stopped once virus was isolated from 1 million PBMCs at 4 consecutive weekly time points.
Administration of cM-T807
CD8+ cells were depleted in a subset of animals (Fig. 1, group D) using the previously described cM-T807 antibody19,20; a total of 30 mg/kg body weight was administered in 3 doses of 10 mg/kg subcutaneously. The first dose was given 3 days before the first oral virus inoculation, with the remaining doses given 3 and 7 days later. Other investigators have previously performed control experiments with nondepleting or B-cell-depleting antibodies to demonstrate that the effects observed on SIV viremia after cM-T807 administration are not due to generalized immune activation.19,21-25
Plasma viral RNA was quantified using a bDNA signal amplification assay specific for SIV, versions 3.0 and 4.0 (which have lower quantitation limits of 500 and 125 copies/mL, respectively).26 Infectious virus was isolated in cultures of PBMCs or lymph node mononuclear cells with CEMxl74 cells and subsequent p27 core antigen measurement via an enzyme-linked immunosorbent assay (ELISA), as described previously.27 Nested PCR analysis to detect proviral DNA in PBMCs and lymph node mononuclear cells was performed using SIV gag-specific primers according to methods previously described.9 Infection was defined as detectable persistent viremia (by viral RNA, infectious virus, or proviral DNA). A few animals had some evidence of early transient low-level viremia (infectious virus in PBMCs, plasma viral RNA, or proviral DNA near the detection limit of the respective assays), but these animals were persistently negative for these criteria afterward and did not have anamnestic SIV-specific antibody responses; such cases of transient viremia or abortive infection are similar to previous observations.9,28
ELISAs to detect immunoglobulin G specific to SIV and other test antigens were performed as described previously.17 Neutralizing antibody titers in plasma were measured according to methods described previously,29 using CEMxl74 cells and laboratory-adapted SIVmac251 (grown in H9 cells). In addition, neutralizing antibody titers were also measured in CEM-R5 cells (ie, CEMxl74 cells expressing CCR5 by trans-fection [which were generously provided by James Robinson]) against the SIVmac251-5/98 challenge stock, which was briefly expanded in human PBMCs; the cutoff value of this assay is a titer of 1:30. Cytokine levels in plasma were measured using commercially available ELISA kits (monkey IL-12 ELISA kits from U-CyTech, Utrecht, the Netherlands; human interferon [IFN]-α ELISA kit from PBL Biomedical Laboratories, Piscataway, NJ).
To estimate the number of SIV-specific IFN-γ-producing T cells in cryopreserved PBMCs, an ELISPOT assay using a pool of 20-mer peptides of the entire p27 Gag region of SIVmac239 was used according to methods described previously10,30; values are reported with subtraction of the values of the medium-control wells. Real-time PCR analysis to quantify cytokine and chemokine mRNA in PBMCs was performed according to methods described previously.31-33 Changes in PBMC mRNA levels in experimental animals are reported compared with average PBMC mRNA levels in un-infected 4-week-old infant macaques. Lymphocyte phenotypic analysis was performed using 3-color and 4-color flow cytometry techniques as described previously.26
Criteria for Killing and Animal Necropsies
Killing of animals with simian AIDS was indicated by criteria described previously.10 A complete necropsy including histopathologic examination was performed on all animals.
The antibody titers and viral RNA levels (plasma SIV RNA copies/mL) were log transformed for all analyses. Kaplan-Meier curves were used to illustrate the survival and infection patterns in the different groups of macaques, and the Wilcoxon log-rank test was used to test for differences in these patterns. For infant macaques that became infected after oral SIV inoculation, virus levels were compared by repeated-measures analysis (from 8 to 22 weeks of age) and the Kruskal-Wallis test for select time points. Cytokine mRNA levels were compared by ANOVA (with the Tukey posttest). For those macaques that were inoculated at juvenile age, virus levels in immunized and unimmunized animals with persistent viremia after SIV inoculation were compared by testing for differences in the mean AUC (area under the time vs. log concentration curve) for the first 6 weeks of viremia using the two-sample t test, with verification of equal variance in the two groups. Assumptions of all tests were verified for these data. Statistical analyses were performed using SAS Version 8.2, Prism Version 4.0 for Mac, and Instat 3 (GraphPad Software, Inc., San Diego, CA), and P < 0.05 was considered statistically significant.
Poxvirus-Based SIV Vaccines Protect Infant Macaques Against Infection: Experimental Design and Summary of Virologic and Clinical Outcome
Infant macaques were immunized with MVA-SIV or ALVAC-SIV within the first 3 weeks of life (Fig. 1). At 4 weeks of age, all animals were fed 15 low doses of SIVmac251. Of 16 unimmunized controls, 14 became persistently viremic (Fig. 2A) compared with 11 of 17 MVA-SIV-immunized animals and 6 of 16 ALVAC-SIV-immunized animals (ALVAC-SIV-immunized animals vs. controls; P = 0.005, one-sided Fisher exact test). All uninfected immunized animals had normal growth and normal clinical parameters throughout the observation period (from 8 to 18 months of age or older; data not shown), suggesting that the MVA and ALVAC vaccines were safe.
As observed in our previous studies with this highly virulent SIVmac251 isolate,9,10,34-36 most unimmunized infant macaques (11 of 14) that became infected had persistently high viremia (>107 RNA copies/mL) and developed simian AIDS within 24 weeks of infection. For the animals that became infected, prior immunization with ALVAC-SIV or MVA-SIV resulted in longer survival (which was statistically significant only for the ALVAC-SIV-immunized group; log-rank test, P = 0.04; Fig. 2C). This prolonged survival was associated with reduced viremia in many immunized animals (average, ∼0.5-1 log lower) compared with the unimmunized animals; however, due to relatively small animal groups with considerable intragroup variability, this difference in viremia was not statistically significant (repeated-measures ANOVA; Fig. 2B).
Because of the promising results with ALVAC-SIV despite its low immunogenicity as measured by in vitro assays (see below), additional animal experiments were performed. To determine the contribution of CD8+ cells to the in vivo effects of the ALVAC-SIV vaccine, 8 animals were immunized using the same regimen of ALVAC-SIV but were depleted of CD8+ cells using the anti-CD8 antibody cM-T807 3 days before the start of the SIVmac251 inoculations. CD8+ cells were efficiently depleted, as demonstrated by the undetectable or low (<0.35%) frequency of CD8+ cells in peripheral blood lymphocytes up to 10 days after the first cM-T807 injection (data not shown). In the absence of CD8+ cells, the ALVAC-SIV vaccine had little protective effect (6 of 8 animals became infected; Figs. 1, 2A, group D). The CD8+ cell-depleted ALVAC-SIV-immunized animals that became infected had plasma viral RNA levels and survival similar to those of the unimmunized animals. Two of 4 animals immunized with ALVAC-vector became persistently infected; viremia and survival were indistinguishable from those of unimmunized animals.
Vaccine-Induced Immune Responses Before SIVmac251 Challenge
Because the overall challenge outcome was similar for ALVAC-SIV- and MVA-SIV-immunized animals and both represent poxvirus vector-based vaccines, a number of immune parameters were evaluated in animals from both experiments to define possible correlates of protection. Blood samples were collected from the 4-week-old infant macaques 3 days before the onset of the low-dose oral SIVmac251 feedings. Immunization with poxvirus-based SIV immunogens resulted in the induction of anti-SIV binding antibodies (measured by inactivated whole-virus ELISA). MVA-SIV-immunized animals had significantly higher titers of SIV-binding antibodies than did ALVAC-SIV-immunized animals (P < 0.001, Mann-Whitney U test) (Fig. 3A). Plasma samples were tested for the presence of neutralizing antibodies; none of the MVA-SIV- and ALVAC-SIV-immunized animals tested had detectable titers of neutralizing antibody to the SIVmac251 challenge virus. Titers of neutralizing antibody to laboratory-adapted SIVmac251 were undetectable or very low (<1:50), except for 7 MVA-SIV-immunized animals that had titers of low magnitude (1:60 to 1:197). However, because 6 of these 7 animals became infected, titers of neutralizing antibody to laboratory-adapted SIVmac251 did not correlate with protection.
Although plasma levels of most cytokines were too low to detect via ELISA techniques, IL-12 and IFN-α were readily detected in plasma samples; however, there was no detectable difference in plasma IL-12 and IFN-α levels between immunized and unimmunized animals at 4 weeks of age, and there was no correlation with subsequent protection from infection with SIVmac251 (data not shown).
SIV Gag-specific IFN-γ lymphocyte responses were measured using an ELISPOT assay. Although no IFN-γ-secreting T cells were detected in ALVAC-SIV-immunized animals at 4 weeks of age, 5 of 9 MVA-SIV-immunized animals tested had detectable but low SIV-specific IFN-γ-secreting T-cell responses (35-100 SFC per 1 million PBMCs; Fig. 3D). The difference in the frequency of SIV Gag-specific IFN-γ-secreting T cells between infected and uninfected MVA-SIV-immunized animals was marginal (P = 0.06, one-tailed Mann-Whitney U test).
In available PBMC samples collected at week 4, mRNA levels were quantified for a panel of proinflammatory cytokines and chemokines (tumor necrosis factor α, macrophage inflammatory proteins α and β, macrophage-derived chemo-kine, type I IFNs [IFN-α and IFN-β], type I IFN-stimulated genes [Mx, PML, and IP-10/CXCL10], and T-helper 1/T-helper 2 response-regulating cytokines [IL-2, IL-4, IL-6, IL-12, and IFN-γ]). In comparison with unimmunized animals, mRNA levels of these cytokines in PBMCs from poxvirus-immunized animals (Fig. 1, groups B-E) were either not significantly or only marginally different (group means were <4-fold different) from those PBMC levels in unimmunized age-matched animals (data not shown). Thus, immunization with these poxvirus-based recombinants within the first 3 weeks of life did not induce any major changes in PBMC cytokine mRNA levels at 4 weeks of age.
In summary, although both MVA- and ALVAC-based recombinants were immunogenic in infant macaques, MVA-SIV was more immunogenic than ALVAC-SIV as measured by SIV-specific antibodies and frequencies of SIV Gag-specific IFN-γ-producing T cells detected in peripheral blood.
Assessment of Immunologic Parameters After Oral SIVmac251 Challenge
Uninfected Infant Macaques
After SIVmac251 inoculation, uninfected animals had no evidence of increased antiviral immune responses. Instead, anti-SIV antibody titers in immunized uninfected animals declined gradually and became undetectable or stabilized at lower levels for the remainder of the 7 months of observation (Fig. 3B). Two MVA-SIV-immunized animals that had detectable SIV-specific IFN-γ-producing T cells at week 4 but did not become infected (Fig. 3D) had similar responses 2 weeks after virus inoculation (55-105 SFC per 1 million PBMCs); however, for samples collected from 4 to 12 weeks after virus inoculation (ie, 8 to 16 weeks of age), SIV-specific IFN-γ-producing T cells were not detected in these 2 uninfected animals (Fig. 3E). Further, cytokine and chemokine mRNA levels in PBMCs did not change significantly. All uninfected animals had normal complete blood cell counts and lymphocyte subset values as well as normal antibody responses to other test antigens (see Methods; data not shown). Thus, there was no evidence of increased SIV-specific immune responses or nonspecific immune activation in animals that were not infected after SIVmac251 challenge.
Infected Infant Macaques
Consistent with previous observations,17,34,37,38 unimmunized animals with high viremia and rapid disease progression had on average low SIV-binding antibody responses (Fig. 3C) and reduced antibody responses to test antigens (tetanus and diphtheria toxoids and cholera toxin B subunit; data not shown). Prior immunization with MVA-SIV and ALVAC-SIV resulted in an anamnestic SIV-binding antibody response after infection (Fig. 3C). SIV-binding antibody titers correlated with titers of neutralizing antibody to laboratory-adapted SIVmac251 (range, undetectable to 1:25,915; P < 0.0001, Spearman r = 0.87), but titers of neutralizing antibody to the SIVmac251 challenge virus remained undetectable or low (<1:90). CD8+ cell depletion in ALVAC-SIV-immunized animals did not alter the kinetics of the anti-SIV antibody response. However, these antiviral antibodies had no detectable beneficial effect on viremia and survival in the absence of CD8+ cells, because these animals had a rapid disease course similar to that of unimmunized animals (Figs. 2, 3C).
Similar to our previous observations in SIVmac251-infected infant macaques,10 SIV-specific IFN-γ-producing T cells were detected infrequently in PBMCs from SIVmac251-infected infant macaques; only 4 MVA-SIV-immunized animals had detectable levels in the first 8 weeks after challenge (45-200 SFC per 1 million PBMCs; Fig. 3E). There was no apparent correlation between the detection of SIV-specific IFN-γ-producing T cells and the disease course: of these 4 MVA-SIV-immunized animals, 2 had rapid disease progression (AIDS within 7 weeks of age) and 2 were among the ones that survived the observation period of 28 weeks.
Because the SIV-specific IFN-γ ELISPOT assays detected few responses, general immune function and activation were also evaluated in PBMCs collected 1 and 2 weeks after SIV challenge (ie, 5 and 6 weeks of age) by quantitation of mRNA levels of a panel of cytokines, chemokines, and chemokine receptors. Interestingly, 2 weeks after SIVmac251 challenge (6 weeks of age), MVA-SIV-immunized SIV-infected infants had significantly higher granzyme and perforin PBMC mRNA levels (1-way ANOVA with Tukey-Kramer multiple comparisons test, P < 0.001) and marginally elevated IFN-γmRNA levels (P = 0.06) compared with unimmunized animals (Figs. 3F-H). This increase in PBMC mRNA levels of cytolytic CD8+ effector molecules in MVA-SIV-immunized animals compared with unimmunized animals after infection is suggestive of vaccine-induced anamnestic antiviral immune responses and is in agreement with the slightly lower virus levels and improved survival in the MVA-SIV-immunized animals. Overall, however, PBMC mRNA levels of granzyme and perforin did not show a correlation with plasma viral RNA levels. There was no apparent difference between vaccinated and unvaccinated SIV-infected monkeys for the other cytokine and chemokine mRNA levels. Instead, increased virus replication in infected animals was associated with increased PBMC mRNA levels of many proinflammatory and/or type I IFN-stimulated cytokines (IL-6, IL-12, IFN-α, MIG/CXCL9, Mx, and IP-10/CXCL10) and the CCR5 chemokine receptor (Pearson correlation, all r values of ≥0.23 and 2-tailed P values of <0.05); thus, these increased mRNA levels were indicative of general inflammation and immune activation. These results are consistent with observations of SIV vaccine studies in adult macaques.32,33
Long-Term Efficacy of Neonatal SIV Vaccines Against Oral Infection
Although anti-SIV immune responses declined in animals that did not become infected after oral SIVmac251 challenges at 4 weeks of age (Figs. 3B, E), we determined whether immunization during infancy could protect against oral exposure to SIV later in life. Six ALVAC-SIV-immunized animals that were uninfected after the repeated low-dose SIV challenges at 4 weeks of age were reinoculated orally at 8 months of age with a relatively high dose of SIVmac251 (2 doses of 105 TCID50). All 6 animals became infected (plasma RNA level of >105 copies/mL at 1 week after challenge); thus, immunization with ALVAC-SIV shortly after birth did not protect against a high-dose oral SIV exposure later in life. Subsequently, to determine vaccine efficacy against a repeated low dose of virus (similar to the first challenge experiments), 20 juvenile animals were challenged weekly with low oral doses of SIVmac251 (see Methods). The 20 juvenile macaques consisted of 5 unimmunized controls (including 4 new juvenile macaques that were never exposed to SIV) and 15 animals that had previously been immunized within the first 3 weeks of life (6 with ALVAC-SIV, 7 with MVA-SIV, and 2 with ALVAC-vector; Table 2). In comparison with the 5 unimmunized animals, animals previously immunized with MVA-SIV or ALVAC-SIV required more exposures to become persistently viremic. After 11 weeks of oral SIV inoculations, only 9 of 15 vaccinated juveniles compared with all unimmunized juveniles were SIV infected (Kaplan-Meier curve log-rank test, P = 0.005; median infection time: 8 weeks for immunized animals vs. 1 week for controls; 71% reduced hazard of SIV infection; Fig. 4A). Animals previously immunized with ALVAC-SIV (n = 6) or MVA-SIV (n = 7) had similar protection against infection (Table 2, Fig. 4A). MVA-SIV- or ALVAC-SIV-immunized animals that became persistently viremic upon rechallenge also had lower viremia than the unimmunized animals (comparison of AUC values: P = 0.02, two-tailed t test; Figs. 4B-D). Thus, immunizations during the first 3 weeks after birth had protective efficacy against oral reexposure to virus ∼1-4 years later.
The goal of this study was to develop an infant macaque model that more closely mimics the many exposures that occur during breast milk transmission of HIV and that allows pre-clinical testing of pediatric HIV vaccine strategies. Although a macaque model has been developed to mimic natural exposure of infants to virus in breast milk (by having SIV-infected female macaques breast-feed their infants),39 direct oral SIV inoculation of infant macaques allows better control of the amount and concentration of the virus inoculum and the timing of exposure. The ability to control such variables is advantageous in testing prophylactic strategies, especially given the limited availability of valuable animals for research.
To our knowledge, the current study demonstrates for the first time that immunization of infant macaques shortly after birth can prevent systemic infection after repeated low-dose oral exposures to virulent SIV. A repeated low-dose exposure model is more representative of human exposure to HIV and may identify effective prophylactic strategies that could be missed using the more common high-dose challenge models (eg, our previous study with MVA-SIV in infant macaques10). Although the virus concentration (7 × 107 RNA copies/mL) that was administered to the 4-week-old infant macaques is higher than the HIV levels in milk of transmitting mothers (median, ∼2-3 × 103/mL; range, <30 to >106/mL),40-42 the large volume of milk consumed by human infants (often >700 mL/d) could potentially provide exposures similar to the daily amounts of virus we used in our SIV challenge model.
The present study demonstrates the prophylactic efficacy of MVA-SIV and ALVAC-SIV (each expressing gag, pol, and env genes) against oral SIV challenge. Approximately 30%-50% lower infection rates were observed for MVA-SIV-and ALVAC-SIV-immunized animals compared with unimmunized animals. These poxvirus-based SIV immunogens were selected because the safety and immunogenicity of similar MVA and ALVAC constructs have been demonstrated in human adults, and pediatric trials are ongoing or planned.5,43 In particular, the HPTN-027 trial proposes testing of an ALVAC-vector based HIV-1 vaccine in breast-feeding infants of HIV-1-infected women in Uganda (www.hptn.org).
An analysis of the immunogenicity of the 2 poxvirus candidates tested in the present study revealed that MVA-SIV was more immunogenic in terms of inducing higher levels of antiviral antibodies and cell-mediated antiviral immunity as measured by SlV-specific IFN-γ-secreting T cells in PBMCs before challenge. Thus, the MVA-SIV vaccine induced several immune responses in infant macaques that are commonly used as predicted surrogate markers of HIV vaccine efficacy to move promising HIV vaccine candidates through clinical trials. Compared with MVA-SIV, ALVAC-SIV was significantly less immunogenic in infant macaques by these standard immune measures. This result is consistent with the relatively low to moderate “immunogenicity” observed for ALVAC-based SIV vaccines in older macaques and in HIV vaccine trials with human adults and infants43-49 (D. Johnson et al, personal communication; E. McFarland et al, personal communication). In particular, the ALVAC vaccine phase 3 correlates trial in the United States was not conducted due to a low frequency of responders with virus-specific IFN-γ-producing cells (by ELISPOT assay) in the earlier safety and immunogenicity trials.50 Nevertheless, ALVAC-SIV-immunized animals had a significantly lower rate of infection after oral SIV exposure than did unimmunized monkeys. Thus, vaccine immunogenicity by the present in vitro measures did not predict the efficacy rate of ∼30%-50% observed in our animal studies and desired in phase 3 HIV vaccine trials. This highlights the limitations of the presently used standard in vitro assays.
Several factors may explain the difficulty in defining more precisely the immune mechanisms of vaccine protection in these infant macaque studies: the animal groups were relatively small; the analysis of immune markers was necessarily limited to peripheral blood, which does not reflect immune activity at the oral mucosa and lymphoid tissues (as demonstrated previously31,33,51); and alternatively, additional immune responses need to be assessed. It is plausible that different vaccines may confer protection through the induction of different antiviral immune responses (or combinations thereof), not all of which may be readily measurable by the current in vitro assays. Despite the low immunogenicity of ALVAC-SIV as measured by in vitro assays, CD8+ cells played a role in the in vivo protection, because CD8+ cell depletion resulted in higher infection rates among ALVAC-SIV-immunized animals and reduced survival for infected animals. Because the cM-T807 antibody depletes both CD8+ T cells and NK cells, the relative contribution of these cell populations to the efficacy of ALVAC-SIV could not be distinguished. In another macaque study, relatively weak SIV-specific immune responses by current standard measures were also able to protect against later viral rechallenge.24 In this context, it is possible that antiviral immune responses that occur during the first hours after mucosal virus exposure (when virus levels are low) are distinct from those immune responses required to curtail virus replication once infection is systemic.52 Further, at low to moderate levels of virus replication, commonly measured antiviral immune responses show a curvilinear relationship with viremia.53,54 The antigenic dependency of these immune responses could explain the lack of detectable anamnestic antiviral immune responses in the uninfected animals in the current study, due to insufficient antigenic stimulation. Thus, there is a need for analysis of immune responses in tissues and a broader analysis of multiple immune parameters. For example, other properties of antiviral antibodies (eg, antibody-dependent cellular cytotoxicity and complement fixation) and cytolytic and noncytolytic antiviral effector functions of CD8+ cells can collectively contribute to antiviral immunity in vivo, but these were not measured. The role of innate immune responses should also be evaluated. Poxviruses have many immunomodulatory and adjuvant properties.55,56 Thus, poxvirus-based vaccines may create an environment of innate and/or adaptive immunity unsuitable for early virus replication and dissemination. Proper innate immune responses can promote the early development of more effective adaptive immune responses that can limit initial virus replication and dissemination.57
An important finding of the present study was that immunization of infant monkeys early after birth still provided protection against oral SIV infection not only at 4 weeks of age but also when challenged as juveniles (∼1.5-4 years of age). This observation suggests that neonatal immunization could reduce the risk of acquiring HIV infection early in life by breast milk transmission but also has the potential to prevent HIV infection through oral sex in the pubertal/adolescent period. Such a neonatal HIV vaccine strategy could be equivalent to the vaccine strategy already used in human infants against hepatitis B aimed at preventing infection later in life. The observation that most immunized animals eventually became infected after continued weekly exposures to oral SIV indicates that an HIV vaccine strategy, even if quite effective in reducing the relative risk of infection per exposure, should still be combined with behavioral risk reduction interventions.
In conclusion, this study demonstrates the utility of a nonhuman primate model with repeated low-dose viral challenge for preclinical testing of candidate HIV vaccines. In particular, the results of the current study suggest that immunization of human newborns with poxvirus-based HIV vaccines may be able to reduce HIV transmission through breast milk.
The authors thank D. Bennett, R. Colón, T. Dearman, L. Hirst, W. von Morgenland, and K. West as well as the Immunology Core, Pathology Staff, Veterinary Staff, Colony Services, and Clinical Laboratory of the California National Primate Research Center for expert technical assistance; Lynn Frampton for construction of recombinant MVAs; the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, for providing the SIV p55 Gag protein and the 20mer peptides of the p24 Gag region; Dr. R. Desrosiers (New England Regional Primate Research Center) for the plasmid containing the SIVmac239 env gene; and Dr. K. Reimann (Beth Israel Deaconess Medical Center, Harvard Medical Center) for providing the cM-T807 antibody (which was produced by the National Cell Culture Center with funds provided by NIH grant RR16001).
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