Experimental design of animal experiments and summary of outcome.
Nineteen newborn rhesus macaques were divided into five experimental vaccine groups (Table ). Group 1 (n = 5) consisted of nonimmunized control animals. One group 1 infant (animal 31608) was born to a pregnant female infected with SIVmac251, and therefore had maternal anti-SIV antibodies, but no virus was detected in this infant at 4 weeks of age. Animals in groups 2 to 5 were immunized at 0 and 3 weeks of age. Group 2 (n = 2), group 3 (n = 4), and group 4 (n = 4) animals were vaccinated with MVA-SIVgpe; group 4 (n = 4) animals had maternally derived SIV antibodies (due to immunization of their mothers with inactived SIV). Group 5 (n = 4) animals were immunized with live-attenuated SIVmac1A11 at 0 and 3 weeks. Except for group 2 (MVA-SIVgpe immunized, but unchallenged infants), all groups were inoculated orally with SIVmac251 at 4 weeks of age. All infants were immunized with cholera toxin subunit B (at 2 and 10 weeks of age) and tetanus toxoid (at 6 and 14 weeks of age) to determine immune responses to nonviral antigens. As described below, the animal groups were compared with regard to a number of parameters (survival, viremia, immune responses, etc.).
Experimental design and disease outcome
SIV infection and disease outcome in infants challenged orally with SIVmac251.
All 17 infants orally inoculated with SIVmac251 (Table , group 1 and groups 3 to 5) became infected 1 week after challenge as determined by virus isolation, bDNA analysis, and reverse transcription-PCR. All unvaccinated SIVmac251-infected animals (group 1) demonstrated a “failure to thrive” syndrome characterized by poor weight gain that was significantly lower than that of normal, uninfected nursery-reared rhesus infants (P < 0.05) (Table ). Four of these five SIVmac251-infected control animals developed life-threatening immunodeficiency within 14 weeks of age, whereas the fifth animal needed euthanasia at 28 weeks of age (Table ). Compared to group 1, the animals in the three immunized, SIVmac251-challenged groups (groups 3 to 5) had significantly longer disease-free survival (Fig. ). Four MVA-SIVgpe-vaccinated animals developed AIDS by 19 weeks (animal 31732), 26 weeks (animals 31533 and 31542) or 27 weeks of age (animal 31526). The remaining eight vaccinated infants, including all four SIVmac1A11-vaccinated animals, were still clinically stable at the time of experimental necropsy (28 weeks of age). The two animals that were vaccinated with MVA-SIVgpe but not challenged with SIVmac251 (group 2) had normal weight gain and remained healthy throughout the observation period (>2 years).
FIG. 1. Comparison of survival for vaccinated and unvaccinated infant rhesus macaques. The survival of unvaccinated (dashed line) and vaccinated (solid lines) animals by age (weeks) is shown. The comparison of survival curves was performed by using the log-rank (more ...) Comparison of viremia in SIVmac251-infected infant macaques.
There was a strong inverse correlation between SIV RNA levels in plasma and disease-free survival. After oral inoculation with SIVmac251 at 4 weeks of age (Table , groups 1 and 3 to 5), SIV RNA levels in plasma peaked 1 to 2 weeks later (Fig. ). For the purpose of further discussions, viral RNA levels were classified as high (>108 RNA copies/ml), moderate (between 107 and 108 RNA copies/ml), or low (<107 RNA copies/ml). For the unimmunized control animals (Table , group 1), virus levels in plasma remained persistently high (four animals) or moderate (animal 31608) until the time of death (Fig. ). In contrast, SIV RNA levels in the immunized infants were lower than in the controls. Although there was individual variation, SIV RNA levels in plasma for the group 3 animals (MVA-SIVgpe immunized) during initial peak viremia (1 to 3 weeks postchallenge) were ca. 10- to 100-fold lower than for the unvaccinated controls (group 1), and at 12 to 16 weeks of age, there were two animals with moderate viremia and two animals with low viremia. Viral RNA levels in group 4 (MVA-SIVgpe immunized with maternal antibodies) during the initial viremia and thereafter were ca. ≤10-fold lower than that observed in the unvaccinated controls, and at 12 to 16 weeks of age the animals had moderate viremia (n = 3) or moderate to high viremia (animal 31732; Fig. ). For the group 5 animals (SIVmac1A11 immunized at 0 and 3 weeks of age), virus could be isolated from the PBMC of two of these infants (animals 31777 and 31780) at 1 and 2 weeks of age, and low levels of viral RNA (53,000 to 80,000 copies/ml) were detected in plasma of three of these infants (animals 31777, 31779, and 31780) at 1 week of age, indicating the presence of replicating SIVmac1A11. After oral inoculation of these animals with SIVmac251 at 4 weeks of age, SIV RNA levels in plasma in three of the group 5 animals initially spiked to levels as high as those observed in the unvaccinated controls (Fig. ), but from 6 weeks of age (2 weeks after challenge) SIV RNA levels declined to 10- to 100-fold lower than those of unvaccinated controls. At 12 to 16 weeks of age, there were two animals of group 5 with moderate viremia and two animals with low viremia, and thus there was no difference in viremia with the group 3 animals.
FIG. 2. Virus levels in blood. Levels of SIV RNA in plasma (top panel) were measured by bDNA assay, whereas cell-associated virus levels (bottom panel) were measured by limiting dilution assay. Vaccine (V) was administered within 3 days after birth and 3 weeks (more ...)
FIG. 3. Measurements of quantitative and qualitative antibody responses in infant macaques orally inoculated with SIVmac251. Quantitative levels of SIV whole virus (A) and gag-specific (B) antibodies were determined in a standard ELISA. SIV Env-specific antibody (more ...)
During the first 10 weeks after oral challenge, each immunized group had significantly reduced mean SIV RNA levels in plasma after SIVmac251 infection compared to the mean SIV RNA levels in plasma in the unimmunized animals (group 3 versus group 1, P = 0.0011; group 4 versus group 1, P = 0.0291; and group 5 versus group 1, P = 0.0019). Due to the small number of animals per group and the larger variation in SIV RNA levels in plasma among vaccinated animals, no statistically significant differences in SIV RNA levels were detected among the three vaccine groups (groups 3, 4, and 5). However, from weeks 8 to 24 after challenge, only vaccinated groups 3 and 5 had mean SIV RNA levels in plasma that were significantly lower than those of the controls (group 3 versus group 1, P = 0.0005; group 4 versus group 1, P = 0.0941; and group 5 versus group 1, P = 0.0012).
Levels of infectious cell-associated SIV in blood of all 17 SIV-infected infants showed considerable variation among animals within each group and temporal variation for individual animals, and thus there were no significant differences among the experimental groups in cell-associated infectious virus levels at the time of peak viremia or thereafter (Fig. ). Cell-associated SIV levels in blood peaked at 1 or 2 weeks after oral challenge (460 to 21,530 TCID50 per million PBMC) and thereafter ranged from 10 to 2,150 TCID50 per million PBMC.
Quantitative and qualitative comparison of antiviral antibody responses in infant macaques.
Total antiviral IgG antibody responses were quantified by whole-SIV ELISA. Gag- and Env-specific antibodies were measured by using protein-based ELISA assays. For those samples with detectable Env-specific antibodies, measurements of antibody avidity and conformational dependence were determined as described in Materials and Methods.
Except for maternally derived antibodies in animal 31608, all five SIVmac251-infected animals of group 1 had undetectable, or else low and transient de novo IgG responses, to whole SIV Gag and Env. The inability of these infants to mount or maintain antiviral antibody responses is a result of the rapid immunosuppression induced by the highly virulent SIVmac251 isolate.
The two animals of group 2, which were immunized with MVA-SIVgpe but were not challenged with SIVmac251, mounted a rapid anti-SIV IgG response by 4 weeks of age (titer of 1:25,600), and the antibodies persisted throughout the observation period (Fig. ). This observation demonstrates the immunogenicity of this vaccine in infant macaques. However, SIV Env-specific endpoint titers were low at 4 weeks of age and became undetectable at 16 to 28 weeks of age. In contrast, SIV Gag-specific antibody responses continued to increase during the 28 weeks. Because MVA has been shown to have limited replication in human cells in vitro (3
), the slow increase in SIV Gag-specific antibody responses in these infant macaques suggest that immunization with MVA-SIVgpe may result in a low level of residual replication with preferential expression of gag
At birth, prior to the immunizations, the four infants of group 4 already had moderate to high levels of maternally derived whole SIV-specific antibodies, moderate levels of SIV Gag-specific antibodies, and except for animal 31732, detectable levels of anti-SIV Env antibodies (Fig. ). At 4 weeks of age (i.e., after the two immunizations with MVA-SIVgpe or SIVmac1A11) all animals of groups 3, 4, and 5 had moderate to high antibody titers against whole SIV (titers of 6,400 to 102,400) but low levels of antibodies to SIV Gag and Env. After oral inoculation with SIVmac251 at 4 weeks of age, antibody titers followed a similar pattern: despite individual variation within each group, the immunized animals of groups 3, 4, and 5 mounted similar levels of whole SIV-, SIV Gag-, and SIV Env-specific antibodies during the first 4 weeks after SIV infection. Later, there was more variation in SIV-specific antibody levels. In general, antiviral antibody titers, in particular the Gag-specific antibody titers, declined for animals that developed AIDS within the 28 weeks of observation period (group 3, animals 31533 and 31542; group 4, animals 31526 and 31732) but remained stable or increased for animals that were clinically still asymptomatic at 28 weeks of age (including all of the group 5, SIVmac1A11-immunized animals). The maternal antibodies in group 4 animals did not have any detectable inhibitory effect on the levels of SIV-specific antibodies produced during the first 4 to 12 weeks of SIVmac251 infection. In summary, the immunizations with MVA-SIVgpe or SIVmac1A11 primed animals in groups 3, 4, and 5 to produce similar levels of SIV-specific antibodies early after SIVmac251 infection.
In contrast, there were marked differences among animals of groups 3, 4, and 5 in the quality of SIV-envelope specific antibodies as assessed by avidity and conformational dependence. In group 3 (MVA-SIVgpe immunized), SIV Env antibodies at the time of oral challenge had low or undetectable avidity but high conformation ratios (indicating reactivity to native envelope glycoprotein). For three of these four animals, avidity index values peaked by 4 weeks after SIV infection but remained low (≤32%) and declined afterward; the conformation ratio in these animals showed a pattern opposite to avidity, in that the conformation ratio dropped sharply 4 weeks after SIV infection but then increased again to higher levels, indicating preferential recognition of native envelope glycoprotein. Animal 31542 was the only animal for which the SIV envelope antibody avidity index increased gradually to intermediate levels (42%) by the onset of AIDS at 26 weeks of age and for which the conformational dependence remained remained high and showed little variation over time.
For the group 4 animals (MVA-SIVgpe-immunized infants with maternally derived SIV antibodies at birth) anti-SIV Env antibodies were detectable in only three of four animals at birth; the anti-env antibodies from these three animals had a moderate avidity index (32 to 45%) and a low conformational dependence (between 1 and 2; Fig. ) that is consistent with a mature antibody response in their immunized mothers (6
). For these three animals, there was a sharp decrease in avidity index after MVA-SIVgpe immunization (i.e., prior to SIVmac251 inoculation). After a further decrease for two animals during the first 4 weeks of SIV infection, there was a slower increase in avidity index compared to group 3 animals (which already had SIV Env antibodies with detectable avidity 4 weeks after oral SIVmac251 challenge; Fig. ). This suggests that the de novo anti-SIV Env antibody response of these infants that was clearly detectable 4 weeks after SIV infection (Fig. ) consisted mainly of antibodies with very low avidity. In contrast, the fourth animal (i.e., animal 31732), which did not have detectable maternally derived anti-env antibodies at birth, mounted anti-env antibodies with moderate avidity within 4 weeks after SIV infection, a finding similar to the response of the group 3 animals that lacked maternal antibodies. For all four animals, the conformational dependence increased after SIV infection but then decreased again for animal 31856.
For the group 5 animals (SIVmac1A11 immunized), SIV-env antibody levels at 4 weeks of age were only detectable at sufficient levels in one animal (animal 31777) to allow measurements of antibody quality; the anti-env antibodies in this animal were found to have low avidity and high conformational dependence (Fig. ). After SIVmac251 infection, SIV-env antibody avidity values for all four SIVmac1A11-immunized animals increased gradually to intermediate levels (30 to 41%) and were higher than those of most of the MVA-SIVgpe-immunized animals of groups 3 and 4. In contrast to the MVA-SIVgpe-immunized animals in groups 3 and 4, the conformational dependence of the anti-SIV Env antibodies of group 5 animals was high (>4) shortly after oral challenge with SIVmac251 but then decreased until 28 weeks of age, a finding consistent with previous reports of antibody maturation resulting from intravenous attenuated SIV infection (6
In summary, there was a correlation between the development and maintenance of high antiviral antibody titers and lower viremia and delayed disease course. In contrast, the time course of the parameters of antibody quality was more complex, and there was no clear correlation of antibody quality with virus levels or survival. The initial mode of exposure to antigen (type of vaccine and the presence or absence of maternal antibodies) appeared to have modulated the parameters of SIV-specific antibody quality early during infection, and the subsequent changes in SIV envelope-specific antibody quality reflect most likely a complex process of antibody maturation in the presence of various degrees of immunosuppression.
Quantification of virus-specific IFN-γ-secreting lymphocytes.
The presence of virus-specific IFN-γ-secreting cells in PBMC was measured by an ELISPOT assay by using stimulation with overlapping peptides of the p24 Gag region. For all animals, cryopreserved PBMC samples that were collected at 4, 5, 6, and 8 weeks of age (i.e., the day of oral SIVmac251 challenge and 1, 2, and 4 weeks afterward) were tested. All PBMC samples collected at the time of the oral SIVmac251 challenge had levels of SIV-specific IFN-γ-secreting cells below the cutoff value (see Materials and Methods). After SIVmac251 infection, only two samples had detectable levels of virus-specific IFN-γ-secreting cells: group 4 animal 31833 (week 8 of age, 80 SFC/million PBMC; medium-control wells, 0 SFC/million PBMC), and group 5 animal 31777 (week 5 of age, 128 SFC/million PBMC; medium control wells, 43 SFC/million PBMC). Both of these animals developed moderate viremia (viral RNA levels at 12 to 16 weeks were between 107 and 108 copies per ml of plasma). None of the animals that developed low viremia (<107) had detectable virus-specific IFN-γ-secreting cells at these early time points after infection. For the two MVA-SIVgpe-immunized animals of group 2, which did not receive SIVmac251 challenge, no IFN-γ-secreting cells were detected at these same time points.
Immune response to cholera toxin and tetanus toxoid.
None of the 19 neonates in the study had any maternal antibodies against cholera toxin at birth. After the first cholera toxin subunit B immunization at 2 weeks of age (2 weeks prior to oral SIV inoculation), all 19 animals developed a strong primary antibody response (cholera toxin-specific IgG titer of ≥25,600 at 8 weeks of age), demonstrating competence of infant macaques to respond immunologically to this antigen. All animals also made an anamnestic response (≥4-fold increase in IgG titer; data not shown) after the second cholera toxin subunit B immunization at 10 weeks of age (6 weeks after oral challenge). These cholera toxin subunit B antibody responses show that even in the unimmunized, SIV-infected control animals (group 1), primary and secondary antibody responses to non-SIV antigens were not impaired at these stages of SIV infection.
Most animals had maternally derived antibodies to tetanus toxoid that declined after birth with a half-life of ca. 2 weeks. At the time of the first immunization (6 weeks of age), tetanus toxoid-specific IgG titers ranged from 1:100 to 1:6,400. For the animals with high anti-tetanus toxoid titers (1:1,600 to 1,601:6,400), an absence of a further decline of tetanus toxoid-specific IgG was therefore interpreted as evidence of a primary immune response. Except for animals 31319 (group 1) and 31526 (group 4), all animals made a primary tetanus toxoid-specific antibody response. Animals received a booster immunization with tetanus toxoid at 14 weeks of age (10 weeks pc). In group 1, only animal 31321 was still alive and made a weak (~2-fold increase in titer) secondary tetanus toxoid response. For groups 2 to 5, there was a correlation between the degree of the booster response with clinical outcome; animals 31732 and 31533 (which both developed AIDS at 19 and 26 weeks of age, respectively) had a weak increase (2- to 4-fold), whereas all other animals had a ≥4-fold increase in tetanus toxoid-specific IgG titers.
Lymphocyte subset populations.
Absolute lymphocyte counts were quite variable over time in all animals, even for animals that were not SIV infected (group 2), and absolute counts of the T- and B-lymphocyte subsets showed similar levels of variability (Fig. ). The percentages of cell types were more reliable parameters for comparing the animal groups.
Lymphocyte subsets in peripheral blood as measured by flow cytometric analyses. The percentages are expressed as fractions of the total number of lymphocytes.
Similar to humans, infant macaques have age-related changes in their numbers of total lymphocytes and lymphocyte subsets (12
). In particular, newborn animals have a high percentage of CD4+
T lymphocytes and a low percentage of CD8+
T lymphocytes, resulting in a high CD4/CD8 ratio (>3). During the first few months of age, there is a gradual decrease in CD4+
T lymphocytes and an increase in CD8+
T lymphocytes (Fig. ), resulting in a decrease of the CD4/CD8 ratio to ca. 2 to 3. There is also an age-related increase in CD20+
B lymphocytes (Fig. ). Lymphocyte subsets of the unchallenged MVA-SIVgpe-immunized animals (group 2) were indistinguishable from those of normal, age-matched control animals. For the other animals, all of which became infected after oral SIVmac251 inoculation at 4 weeks of age, some interesting observations were made. Except for the SIVmac1A11-immunized animals, the majority of the other animals had at least a 35% drop in absolute lymphocyte counts at 1 week postchallenge. Whereas the unimmunized SIVmac251-infected animals showed absolute counts and percentages of CD20+
B lymphocytes near or below the range for uninfected animals, most immunized animals (groups 3 to 5), in particular the SIVmac1A11-immunized infants (group 5), had B lymphocytes within the normal range.
Unexpectedly, the unimmunized SIV-infected animals (group 1) showed percentages and numbers of CD4+ T lymphocytes that were generally in the range of the uninfected animals (Fig. ). In contrast, most animals of groups 3, 4, and 5 had a sudden decrease in percent CD4+ T lymphocytes 2 weeks after SIVmac251 inoculation, which slowly recovered but did not reach preinfection baseline values. Absolute CD4+-T-lymphocyte counts in groups 3 to 5 were within the normal range (>1,000/μl) during the first few months after SIV infection, but total lymphocyte counts were also often elevated (Fig. ). The numbers of CD4+ T lymphocytes declined in seven animals of groups 3 to 5 by ≥16 weeks of age to <1,000/μl, concomitant with a decline in absolute total lymphocyte counts.
For the majority of animals, SIV infection was associated with increased and more variable percentages of CD8+ T lymphocytes. An exception were the SIVmac1A11-immunized animals (group 5) and the MVA-SIVgpe-immunized animals of groups 3 and 4 that had slower disease progression (e.g., animals 31540 and 31856), for which the percentage of CD8+ T lymphocytes showed a minor increase after SIVmac251 infection and remained more stable throughout the observation period (Fig. ).
As a result of these opposite changes in the percentages of CD4+ and CD8+ T lymphocytes, the CD4/CD8 ratios were essentially unchanged in most of the unimmunized SIVmac251-infected animals (group 1) but were reduced in all immunized SIVmac251-infected groups (groups 3 to 5), especially in the MVA-SIVgpe-immunized animals (groups 3 to 4), with a nadir occurring 2 weeks after SIVmac251 infection (Fig. ).