SIVmac251 and SIVsmE660 differ by typical intraclade HIV-1 distance.
To evaluate the genetic relatedness of two isolates of SIV that are frequently used in nonhuman primate studies, we compared the genetic distance between SIVmac251 and SIVsmE660 to intraclade and interclade HIV-1 sequence distances. We used HIV clade B and C sequences in the Los Alamos HIV Sequence Database to generate our estimates of HIV-1 interclade and intraclade diversity. We used one sequence per person for these alignments. We analyzed 11,484 pairs of sequences for gag, 21,177 pairs of sequences for env, 32,465 pairs of sequences for nef, and 7,140 pairs of sequences for pol. Figure shows the distribution of normalized frequencies for percent similarity of intraclade and interclade pairwise comparisons. The calculated distances between SIVsmE660 and SIVmac251 at gag, pol, env, and nef are plotted in each panel. As shown in Fig. , the distance between gag and env of the two SIV strains is similar to HIV-1 clade B and C intraclade distances, with distances of 0.91 and 0.83, respectively. In contrast, the distances between the two SIV isolates in pol and nef are of the magnitude seen in interclade differences in HIV-1 (Fig. ). Therefore, these two pathogenic SIV isolates are well suited for use in an SIV model of superinfection because their two key foci, env and gag, have differences that reflect a degree of sequence heterogeneity comparable to different circulating HIV-1 isolates within the same clade.
FIG. 1. Genetic distances between SIVmac251 and SIVsmE660 in relation to HIV-1 clade B and C intraclade and interclade distances. We performed pairwise comparisons of 11,484 gag (A), 21,177 env (B), 7,140 pol (C), and 32,465 nef (D) sequences from individuals (more ...) Plasma SIV RNA levels following primary infection.
We then established cohorts of rhesus monkeys that were infected with one or the other of these two strains of SIV. The viruses and routes of administration used to initiate these infections are summarized in Table . Eight animals were initially infected with SIVmac251 (Fig. ), and six animals were initially infected with SIVsmE660 (Fig. ). Infection was successfully established in 9 of these 14 monkeys via the intrarectal route. However, 5 of 14 monkeys did not exhibit detectable viremia after 18 sequential intrarectal inoculations and had to be inoculated intravenously to initiate the primary infection (CR53, AV74, and CG5G with SIVmac251 and CR54 and CP37 with SIVsmE660).
Viruses, routes of infection, viral load, and time of superinfection
FIG. 2. Plasma viral RNA levels following primary infection with either SIVmac251 or SIVsmE660. (A) Six rhesus monkeys were infected with SIVmac251, and (B) eight were infected with SIVsmE660 via either intrarectal (IR) or intravenous (IV) inoculations. Although (more ...)
Viral replication during primary infection occurred with kinetics typical of SIV replication in naïve rhesus monkeys. Moreover, SIV replication kinetics did not differ significantly between animals that became infected by mucosal or intravenous routes. Monkeys that were infected with SIVmac251 all developed uniform peak plasma viral RNA levels of 6 to 7 logs at 14 days after virus inoculation followed by a sustained viremia of 4 to 6 logs of plasma viral RNA, with the exception of one monkey (CT76) who had undetectable viremia by 700 days postinfection.
In the cohort of monkeys infected by SIVsmE660, monkeys had peak plasma viral RNA levels of 5 to 8 logs at 14 days after virus inoculation, followed by sustained viremia of 5 to 7 logs of plasma viral RNA in animals CP37 and CP23. However, three of the monkeys infected with SIVsmE660 (CP3C, CG7G, and AK9F) had undetectable plasma viral RNA levels by 700 days postinfection, while monkey CR54 had an undetectable plasma viral RNA levels by 85 days postinfection. This wide range in peak and set point viremias in monkeys infected with SIVsmE660 has been previously described (7
). Since plasma viral RNA levels at peak and set point in some of the SIVsmE660-infected monkeys (CP37, CP23, and CG7G) were of a magnitude comparable to that seen in monkeys following SIVmac251 infection, the variability in SIVsmE660 replication levels in monkeys likely reflects a host factor effect rather than an intrinsic lack of replicative capacity of the SIVsmE660 strain.
Plasma SIV RNA levels following superinfection.
Once set point plasma virus RNA levels were reached, all monkeys were exposed to the heterologous virus by 6 weekly intrarectal inoculations. The duration of primary infection and plasma virus RNA levels at time of exposure to the second virus are summarized in Table . The eight SIVmac251-infected monkeys and six SIVsmE660-infected monkeys were then monitored for evidence of superinfection by assessing plasma SIVmac251 and SIVsmE660 RNA weekly for 20 weeks.
To monitor the viral replication dynamics for each SIV strain in the dually infected monkeys, we developed a qRT-PCR assay using strain-specific probes. Figure shows the replication kinetics of the two strains of SIV following the first and second infections. As depicted in Fig. , six of six monkeys that were initially infected with SIVsmE660 became superinfected with SIVmac251. Of the eight monkeys that were initially infected with SIVmac251, six became superinfected with SIVsmE660 (Fig. ). Viral RNA of the heterologous SIV strain was detected by 14 to 21 days after challenge. In 11 of 12 superinfected animals, with the exception of AK9F, the levels of plasma viral RNA of the second virus at peak viremia were 1 to 4 logs lower than the peak viremia of the first virus. In addition, the levels of plasma viral RNA of the second virus also declined rapidly to undetectable levels in six animals (CR54, CP23, CR53, PBE, AH4X, and CG71), while the viral load persisted at low levels in the remaining six animals (CP37, CG7G, CP3C, AK9F, CP1W, and CT76). The presence of the superinfecting virus at multiple time points was confirmed in each animal by direct sequencing.
FIG. 3. Plasma viral RNA levels of both SIV strains following the primary infection and superinfection in each monkey. Monkeys were either first infected with SIVsmE660 and then with SIVmac251 (A) or first infected with SIVmac251 followed by SIVsmE660 (B). Only (more ...)
Of the 14 infected animals that were exposed to a heterologous virus, only 2 (AV74 and CG5G) that were initially infected with SIVmac251 resisted superinfection with the heterologous virus (Fig. ). There was no detectable SIVsmE660 viral RNA in these animals for 20 weeks after exposure. The absence of replication by the second virus was verified by direct sequencing (data not shown).
No apparent acceleration in disease progression after superinfection.
Interestingly, we observed an increase in plasma viral RNA levels of the primary virus (Fig. ) and a transient decline in CD4+
T cells following superinfection in all of the animals, except AH4X (Fig. ), CP3C, and AK9F (Fig. ). This finding is consistent with case reports of HIV superinfection in which superinfected individuals developed a transient perturbation in total plasma viral RNA levels in association with a clinical prodrome that aroused suspicion that an intervening event might have caused a sudden rise in viral load (2
). The CD4+
T-cell counts re-equilibrated 2 to 6 weeks after superinfection, and a small increase in the CD4+
T-cell counts in some of the animals was observed from 42 to 126 days after superinfection (CT76, CP1W, CG71, CP3C, AK9F, and CG7G). We did not perform statistical analyses on the differences in the CD4+
T-cell decline between superinfected and nonsuperinfected animals due to the small sample size of animals that resisted superinfection, but the trends in changes of CD4+
T-cell counts were indistinguishable between all animals. Therefore, there appeared to be no acceleration in disease progress in the superinfected monkeys as a consequence of superinfection.
FIG. 4. Absolute CD4+ T-cell counts for 126 days after superinfection. The CD4+ T-cell counts in the peripheral blood are shown in blue for the six animals that were initially infected with SIVmac251 and then superinfected with SIVsmE660 (A), (more ...) Peak viral replication following the second infection was lower than peak viral replication following the first infection.
Of the 12 monkeys that became superinfected, 11 animals efficiently controlled the second virus at peak viremia, with the exception of AK9F. Peak replication following the second virus infection was lower than peak replication after the first infection in each monkey (Fig. ). The decrease in peak viremia was statistically significant as determined by the paired Wilcoxon rank sum test (P = 0.001). Furthermore, when considered as a cohort, the median peak viral load value following the second infection was lower than that observed following the first infection (Fig. ). The difference in the median values and interquartile ranges of peak viremia between the first and second infections was statistically significant as determined by the unpaired Mann-Whitney U test (P < 0.0001).
FIG. 5. Peak plasma viral RNA levels were higher following the first infection than after the second infection. (A) Peak plasma viral RNA levels for each monkey following primary infection and superinfection are indicated by individual filled circles and are (more ...) Susceptibility to superinfection was not associated with time after the first infection or persistence of the primary virus.
In these two cohorts of monkeys, superinfection was initiated between 3 and 20 months after the primary infection (Table ). This large window of susceptibility suggests that infected individuals are likely susceptible to superinfection regardless of the state of immune competence of the host or the maturity of the immune response to the initial virus. Superinfection can occur after the immune response against the initial infection has had time to develop and mature. In addition, since 10 of 12 superinfected animals harbored the Mamu-A*01, -B*08, and -B*17 alleles (Table ), susceptibility to superinfection appears not to be a consequence of major histocompatibility complex alleles that are associated with relatively efficient viral control.
Furthermore, the likelihood of acquiring a second virus appears not to be correlated with the persistence of replication of the primary virus at the time of exposure to the heterologous virus (Table ). Some animals became superinfected despite relatively high levels of replication of the primary virus, ranging from 104 to 106 RNA copies/ml in the plasma (CP23, CP37, CP1W, PBE, CG71, AH4X, and CR53), while others became superinfected in the setting of undetectable or low-level replication of the primary virus, ranging from 102 to 103 RNA copies/ml in the plasma (CP3C, CG7G, AK9F, CR54, and CT76).
Interestingly, in animals that had a high-set-point viremia following exposure to the first virus, either SIVmac251 (CP1W, CR53, PBE, AH4X, and CG71) or SIVsmE660 (CP37 and CP23), the second virus was efficiently controlled after superinfection while the first infecting virus remained the predominant viral quasispecies in the plasma. In contrast, in animals that had undetectable plasma viral RNA levels following exposure to SIVsmE660 (CG7G, CP3C, and AK9F) or SIVmac251 (CT76) prior to superinfection, the heterologous virus replaced the first viral strain after superinfection even in monkeys with blunted peak replication of the second virus. Only one monkey in the cohort, CR54, was able to control both viruses to undetectable levels. These data suggest that, although direct viral interference did not contribute to susceptibility to superinfection, it may have influenced the viral replication dynamics of the second virus relative to the primary virus after superinfection.
Susceptibility to superinfection was not associated with absolute CD4+ T-cell counts or percent central memory CD4+ T cells.
To determine if there were any clinical parameters associated with relative susceptibility to superinfection in these cohorts of monkeys, we assessed the absolute CD4+ T-cell counts and the percentage of CD4+ T lymphocytes that were central memory cells immediately prior to the exposure of these animals to the heterologous virus. There was no difference between absolute CD4+ T-cell counts or the percentage of CD4+ central memory T cells in the animals that became superinfected and those that resisted superinfection (Fig. ). Although a statistical analysis could not be performed to validate this observation due to the small sample size of animals that resisted superinfection, the absolute CD4+ T-cell counts and the percentage of central memory CD4+ T cells of animals that resisted superinfection were within the range of the corresponding parameters in animals that became superinfected. In addition, we also analyzed the percentages of effector and naïve memory CD4+ T cells and found that there were no differences in these values between the two groups of monkeys (data not shown). Together, these data indicate that animals with immune systems that are more damaged by a prior SIV infection appeared not to have an increased susceptibility to superinfection.
FIG. 6. Resistance to SIV superinfection was not associated with peripheral blood absolute CD4+ counts or central memory CD4+ T cells at the time of exposure to the superinfecting virus. (A) CD4+ T-lymphocyte counts on the day of challenge (more ...) Susceptibility to superinfection was not associated with virus-specific cellular immune responses.
To determine whether systemic virus-specific cellular immune responses conferred protection against heterologous virus in the monkeys that resisted superinfection, all rhesus monkeys were evaluated for SIV-specific cellular immunity immediately prior to exposure to the heterologous virus. Cellular immunity to SIV was first evaluated using an ELISPOT assay to assess PBMC IFN-γ responses following exposure to a pool of SIV Gag peptides (Fig. ). SIV-specific T-cell responses were indistinguishable between the animals that became superinfected and those that resisted superinfection.
FIG. 7. Resistance to superinfection was not associated with SIV Gag-specific CD4+ and CD8+ T-lymphocyte responses at the time of exposure to the superinfecting virus. Peripheral blood lymphocytes obtained from the monkeys prior to challenge with (more ...)
SIV-specific CD4+ and CD8+ T-lymphocyte function was further evaluated by intracellular cytokine staining. Immediately prior to exposure to the heterologous virus, PBMC production of IFN-γ, TNF-α, and IL-2 was assessed after stimulation with SIV Gag peptide pools. We were able to detect virus-specific CD4+ (Fig. ) and CD8+ (Fig. ) T-lymphocyte responses in PBMCs of all monkeys. We did not perform statistical analyses on the differences in cytokine secretion between the two groups of monkeys due to the small sample size of animals that resisted superinfection. However, the cytokine responses of the two animals that resisted superinfection were within the range of the corresponding parameters in animals that became superinfected. Therefore, the qualitative and quantitative cell-mediated SIV-specific immune responses of monkeys that became superinfected and those that resisted superinfection appeared to be indistinguishable. These findings suggest that SIV-specific cellular immune responses likely did not account for the variability in the susceptibility of these monkeys to superinfection.
Antibody responses did not protect against superinfection.
The role of neutralizing antibody responses in protecting against HIV superinfection is not clear (5
). To assess whether SIV-specific antibodies played a role in the resistance to superinfection in these cohorts of animals, plasma samples harvested just prior to the heterologous viral challenge were assayed for neutralizing antibody responses elicited by the primary SIV infection. The ability of plasma antibody to neutralize SIVsmE660 and SIVmac251 was measured in luciferase reporter gene neutralizing antibody assays using uncloned SIVsmE660 and pseudoviruses expressing viral Envelope cloned from SIVmac251CS.41 (33
). The serum ID50
neutralizing titers against both viruses are shown in Table , Plasma from five of six monkeys (except CR54) that were first infected with SIVsmE660 neutralized the homologous SIVsmE660 (1:62 to 1:508), while plasma from five of eight SIVmac251-infected monkeys neutralized homologous SIVmac251 (1:33 to 1:215).
Neutralizing antibodies in rhesus animals after primary infection prior to superinfection
To investigate whether the antibodies generated by these animals following primary infection have the ability to neutralize the heterologous virus, we assayed the plasma of the monkeys for neutralization activity against the second virus before their exposure to that virus. As shown in Table , animals initially infected with SIVsmE660 generated undetectable or low titers of neutralizing antibodies to SIVmac251 (ranging from undetectable to 1:41). We also detected neutralizing antibodies against SIVsmE660 in six of eight animals that were initially infected with SIVmac251 (ranging from 1:73 to 1:245). The titers against the heterologous SIVsmE660 in the SIVmac251-infected animals were not significantly lower than the titers against the homologous SIVsmE660 in SIVsmE660-infected animals (P = 0.95, Mann-Whitney test).
Interestingly, animals AV74 and CG5G, who were initially infected with SIVmac251 and subsequently resisted superinfection with SIVsmE660, had neutralizing antibodies against SIVsmE660 prior to exposure to this heterologous virus. However, the titers of these antibodies were within the range of antibody titers against SIVsmE660 that were generated by other SIVmac251-infected animals that became superinfected following exposure to SIVsmE660. We did not perform statistical analyses of the differences in antibody titers against SIVsmE660 between the SIVmac251-infected monkeys that resisted superinfection and the SIVmac251- infected monkeys that became superinfected because of the small number of animals that resisted superinfection. Nevertheless, the titers of neutralizing antibodies specific for the heterologous viruses that were elicited during primary infection appear to not have influenced the susceptibility of monkeys to superinfection.