INTRODUCTION

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Perinatal infection with human immunodeficiency virus (HIV) occurs in about 20 to 40% of infants born to HIV-infected women. Although transmission can occur in utero and postnatally (through breastfeeding), evidence suggests that a large fraction of infants become infected during birth by contact with maternal blood and fluids, presumably by an oral route of infection (2, 50).

Pediatric AIDS Clinical Trials Group (ACTG) Protocol 076 demonstrated that zidovudine (ZDV) administration to HIV-infected pregnant women and their newborns can reduce the rate of vertical transmission by two-thirds (6). Because ZDV therapy has little effect on maternal virus levels and ZDV is effective in reducing transmission regardless of the maternal HIV-1 RNA copy number in plasma, it is believed that a significant portion of the protection is due to chemoprophylaxis in the newborn following exposure to the virus, rather than to a reduction of virus levels in the maternal blood (3, 12, 27, 28, 38). Further evidence for this chemoprophylactic mechanism was provided by a recent study in Uganda which demonstrated that the rate of perinatal HIV transmission was greatly reduced by administration of two doses of nevirapine, the first given to the mother in labor and the second given to the infant shortly after birth (16). Because neither the ZDV nor the nevirapine regimen is 100% effective yet, trials are currently evaluating the potential of additional antiviral drugs to further reduce vertical HIV transmission.

Prolonged drug treatment of HIV-infected patients, including pregnant women, usually results in incomplete suppression of virus replication and the emergence of drug-resistant viral mutants. Numerous studies have focused on trying to determine how these drug-resistant HIV mutants affect the efficacy of chemotherapy for patients with chronic HIV infection (18). It is, however, unclear how the drug susceptibility of the virus affects the success of chemoprophylactic strategies aimed at preventing vertical transmission, when newborns are being exposed mucosally to a low dose of virus. For instance, although vertical transmission of HIV-1 resistant to ZDV or other antiviral drugs has been described in a few cases (4, 14, 36; V. A. Johnson, C. Woods, C. D. Hamilton, and S. A. Fiscus, Abstr. 6th Conf. Retroviruses Opportunistic Infect., abstr. 266, 1999; B. Masquelier, E. Lemoigne, I. Pellegrin, D. Douard, B. Sandler, and H. J. A. Fleury, Letter, N. Engl. J. Med. 329:1123-1124, 1993), due to limited data, it is still unclear to what extent ZDV resistance would translate into a reduced efficacy of the ACTG 076 regimen (10, 22). To better understand how drug resistance may affect perinatal HIV transmission during antiviral drug administration, one must evaluate the contribution of a number of variables that may affect vertical HIV transmission, such as the amount of drug-resistant virus that is present in the maternal blood or mucosal secretions, the level of resistance (low or high), the virulence, and the efficiency of drug-resistant viral mutants to transmit across mucosal barriers. Accordingly, it has been difficult to define the specific role of drug susceptibility in the likelihood of vertical HIV transmission and the success rate of chemoprophylactic strategies. An animal model can allow dissection of these variables, because it allows controlled approaches which would be logistically or ethically difficult to test in humans, such as direct inoculation of animals with drug-resistant virus.

Simian immunodeficiency virus (SIV) infection of newborn and infant rhesus macaques is a very useful animal model of pediatric AIDS to rapidly evaluate the efficacy of therapeutic and prophylactic intervention strategies and to define the virulence of drug-resistant viral mutants (43, 46, 49). We have previously demonstrated that short-term administration of the inhibitor of reverse transcriptase (RT) 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir) is highly effective in protecting newborn macaques against oral infection with virulent wild-type SIVmac251 (41, 47). We have also demonstrated that prolonged PMPA treatment of infant macaques with established SIVmac251 infection results in the emergence of SIV mutants with fivefold reduced in vitro susceptibility to PMPA, associated with the development of a lysine-to-arginine mutation at amino acid 65 (K65R) and additional changes in RT; because these additional substitutions did not further reduce the susceptibility of the virus to PMPA, they probably represented compensatory mutations (43). We further studied the pathogenesis of two K65R SIVmac isolates, SIVmac055 and SIVmac385, which have similar fivefold-reduced in vitro susceptibility to PMPA but different additional substitutions in RT (44). Following intravenous inoculation of newborn macaques, both K65R isolates were found to be fully virulent. Replication of the SIVmac055 isolate (K65R, N69T, R82K A158S, and S211N), however, was the most difficult to inhibit in vivo by PMPA treatment, since PMPA administration starting 3 weeks after virus inoculation failed to suppress viremia in SIVmac055-infected infant macaques; however, PMPA therapy still had therapeutic benefits by prolonging survival (44). In the present study, we chose the fully virulent SIVmac055 isolate to inoculate newborn macaques orally and to investigate how a fivefold-reduced in vitro susceptibility of the virus to PMPA affects the ability of PMPA to prevent infection. We demonstrate that short-term prophylactic PMPA treatment protected some newborn macaques against SIVmac055 infection, while the animals which became infected despite short-term PMPA administration had a delay in viremia and prolonged survival compared to untreated SIVmac055-infected infant macaques.

	MATERIALS AND METHODS

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Animals, virus, and PMPA administration. Newborn rhesus macaques (Macaca mulatta) were from the type D retrovirus- and SIV-free colony at the California Regional Primate Research Center and were hand-reared in a primate nursery in accordance with American Association for Accreditation of Laboratory Animal Care Standards. We strictly adhered to the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (31). When necessary, animals were immobilized with ketamine HCl (Parke-Davis, Morris Plains, N.J.) at 10 mg/kg, injected intramuscularly.

Quantitative virus isolation (cell associated and cell free). Levels of infectious virus in cells and plasma of peripheral blood were determined regularly by a limiting-dilution assay (four replicates per dilution) of PBMC and plasma, respectively, in cultures with CEMx174 cells in 24-well plates and subsequent p27 core antigen measurement, by using previously described methods (48-50). In addition, for animals with low or undetectable virus load, 1 × 10⁶ to 5 × 10⁶ PBMC were cocultivated for 8 weeks with CEMx174 cells in tissue culture flasks (48). Virus levels in fresh lymphoid tissues (lymph nodes, spleen, and thymus) collected from the animals at the time of euthanasia were determined by aseptically teasing tissues into single-cell suspensions of mononuclear cells and performing a limiting dilution culture assay similar to the one described above for PBMC. For animals which were not euthanized, an axillary lymph node was recovered by transcutaneous biopsy.

Viral RNA levels in plasma. Quantitative assays for the measurement of SIV RNA were performed by a branched-DNA signal amplification assay specific for SIV (P. J. Dailey, M. Zamroud, R. Kelso, J. Kolberg, and M. Urdea, Abstr. 13th Annu. Symp. Nonhuman Primate Models AIDS, abstr. 99, 1995). This assay is similar to the Quantiplex HIV RNA assay (33), except that target probes were designed to hybridize with the pol region of the SIVmac group of strains including SIVmac251. SIV pol RNA in plasma samples was quantified by comparison with a standard curve produced using serial dilutions of cell-free SIV-infected tissue culture supernatant. The quantification of this standard curve was determined by comparison with purified, quantified, in vitro-transcribed SIVmac239 pol RNA. The lower quantification limit of this assay was 10,000 copies of SIV RNA per plasma sample. Due to the limited blood volume that can be collected from newborn macaques, plasma volumes of 50 µl were available during the early time points, which limited the sensitivity of this assay to 200,000 copies of SIV RNA per ml of plasma.

PCR amplification. Nested PCR was carried out in a GeneAmp 9600 thermocycler (Perkin-Elmer Cetus, Emeryville, Calif.). Two rounds of 30 cycles of amplification were performed on aliquots (at 5 or 10 replicates per sample) of PBMC or mononuclear cells of lymphoid tissues by using SIVmac-specific gag primers and conditions described elsewhere (25). Positive controls included PBMC lysates from known SIV-infected animals. To detect potential inhibitors of Taq polymerase in cell lysates, -actin DNA sequences were amplified by two rounds of PCR (25).

Anti-SIV class-specific antibody determination. The anti-SIV IgG- and IgM-specific antibody ELISAs have been described previously (32, 47).

T-lymphocyte phenotyping. T-lymphocyte antigens were detected by direct labeling of whole blood with PerCP-conjugated anti-human CD8 (Leu-2a; Becton Dickinson Immunocytometry Inc., San Jose, Calif.), phycoerythrin-conjugated anti-human CD4 (OKT4; Ortho Diagnostic Systems Inc., Raritan, N.J.), and fluorescein-conjugated anti-human CD3 (Pharmingen, from Becton Dickinson). Red blood cells were lysed, and the samples were fixed in paraformaldehyde by using the Coulter Q-prep system (Coulter Corp., Hialeah, Fla.). Lymphocytes were gated by forward and side light scatter and were then analyzed with a FACScan flow cytometer (Becton Dickinson).

Drug susceptibility assay. Phenotypic drug susceptibility of SIVmac isolates was characterized by an assay which was used previously to detect SIV mutants with decreased susceptibility to PMPA and other antiretroviral drugs (43, 44, 49).

Sequencing of the viral RT region. CEMx174 cells infected with virus isolated from the SIV-infected animals were harvested as soon as culture supernatants were positive by antigen capture ELISA (24). The genomic DNA preparation, PCR, and sequencing of the RT region were done by previously described methods (43, 44). In brief, PCR was performed on each DNA sample in at least two separate reactions, and the PCR products were subjected directly to dideoxy-DNA sequencing reactions followed by automated analysis by the ALFexpress DNA analysis system (Pharmacia, Piscataway, N.J.). Sequence data were aligned using the DNA STAR software program. The RT amino acid sequence was compared with that of uncloned SIVmac251; the RT sequence of uncloned SIVmac251 was identical to that reported for the molecular clone SIVmac251 (30) with the exception of alanine instead of threonine at position 11. From plasmid-mixing experiments, it was determined that this sequencing method can detect the presence of a 20% subpopulation in the PCR mixture.

Necropsy of the animals and collection and preparation of tissue samples. Euthanasia of animals with simian AIDS was indicated by three or more of the following clinical observations: weight loss of >10% in 2 weeks or >30% in 2 months; chronic diarrhea unresponsive to treatment; infections unresponsive to treatment; inability to maintain body heat or fluids without supplementation; persistent, marked hematologic abnormalities, including lymphopenia, anemia, thrombocytopenia, or neutropenia; and persistent, marked splenomegaly or hepatomegaly (26). A complete necropsy examination was performed on all animals, and a routine histopathologic examination was done on tissues collected at necropsy.

Statistical analysis. Statistical analysis was used to compare PMPA-treated and untreated SIV-infected animals with regard to survival and virus levels. Survival was compared by the generalized Wilcoxon test (8). Virus levels in peripheral blood and development of antibody responses were compared by calculating the area under the curve for each animal for the first 6 weeks after SIV inoculation, followed by analysis according to the Wilcoxon rank-sum test (8). We have previously shown that these analyses can distinguish biologically relevant differences (26, 44, 45, 49).

	RESULTS

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Study design. Within 3 days of birth, eight newborn macaques were inoculated orally with SIVmac055 (Table 1). One group of three animals consisted of untreated SIVmac055-infected control animals (Table 1, group A). Starting 24 h before oral SIVmac055 inoculation, the other group of five animals was given PMPA treatment (30 mg/kg, administered subcutaneously once daily). The PMPA treatment was continued for 4 weeks (Table 1, group B). To monitor the immune response to nonviral, nonreplicating antigens, all newborn rhesus macaques were also immunized with cholera toxin B subunit subcutaneously prior to the SIV inoculation and with a booster immunization at 8 weeks of age.

Control newborn macaques infected with SIVmac055. The three untreated control animals became infected following oral SIVmac055 inoculation and maintained persistently high virus levels in peripheral blood (Fig. 1). One of these three untreated SIVmac055-infected animals (animal 30304), despite making a detectable anti-SIV IgM response (titer 1:200 at 2 weeks [data not shown]), failed to make a detectable anti-SIV IgG response. The other two untreated SIVmac055-infected infants developed a detectable anti-SIV IgG response, but this antibody response was either weak or transient (animals 30169 and 30307, respectively [Fig. 1]). Following cholera toxin B subunit immunization at birth, animal 30304 did not make a detectable IgG response to this antigen while animals 30169 and 30307 made a strong antibody response (titer of 1:102,400 at 6 to 8 weeks of age [data not shown]). These three untreated SIVmac055-infected infants had poor weight gain (Table 1); they developed clinical signs consistent with progressive immunodeficiency and were euthanized between 6 and 12 weeks of age. Similar to previous observations with SIVmac251-infected infant macaques that show a rapid, fulminant disease course (42), CD4/CD8 T lymphocyte ratios and absolute CD4 T lymphocyte counts in peripheral blood of these SIVmac055-infected infants were rather variable and were not always reduced at the time of euthanasia (Table 2). These three untreated control infants had widespread systemic dissemination of virus in lymphoid tissues (Table 2), and the histopathological findings were consistent with terminal SIV infection in this age group (Table 3).

View larger version (44K): [in this window] [in a new window]   FIG. 1.   Time course of SIVmac055 infection of newborn macaques and the therapeutic effects of PMPA. All newborn macaques were inoculated at birth orally with SIVmac055. The three untreated SIVmac055-infected infant macaques are indicated by solid lines. The dashed lines represent the SIVmac055-inoculated animals that became infected despite four weeks of PMPA treatment starting 1 day prior to virus inoculation (shaded area). The animals which did not become infected had undetectable virus and antibody levels and are not indicated on the graphs for purposes of clarity. Infectious virus levels in PBMC (A) and plasma (B) were determined by limiting-dilution culture of PBMC and plasma, respectively. RNA levels in plasma (C) were measured by the branched-DNA assay; IgG titers to SIV (D) were determined by ELISA and are expressed as the reciprocal of the highest of fourfold dilutions (starting from a 1:100 dilution, with two replicates per dilution) which gave a positive optical density above cutoff value. Euthanasia because of simian AIDS is indicated (+).

Newborn macaques inoculated with SIVmac055 and receiving short-term PMPA administration. For two SIVmac055-inoculated animals (30162 and 30306) which received PMPA treatment for 4 weeks, no evidence of infection could be detected; these two animals were tested repeatedly during an observation period of 14 to 28 months. No infectious virus could be isolated, and no proviral sequences could be detected by PCR in PBMC from these two infants. In addition, no SIV-specific antibodies (IgM or IgG) could be detected in plasma samples from these two animals as measured by ELISA and immunoblotting (data not shown). These two animals had normal CD4⁺ and CD8⁺ T-lymphocyte counts and CD4⁺/CD8⁺ T-lymphocyte ratios throughout the 14 to 24 months of observation, and they made good and long-lasting antibody responses to cholera toxin B subunit following immunizations with this antigen at birth and 8 weeks of age (cholera toxin-specific IgG titers for both animals were 1,600 and >102,400 at 8 and 12 weeks of age, respectively). In addition, these two animals had rapid weight gain (similar to uninfected infants at the California Regional Primate Research Center [Table 1]) and were healthy throughout their observation period. Animal 30306 was euthanized at 14 months of age for a more thorough analysis of lymphoid tissues: no infectious virus or proviral DNA could be detected in the peripheral blood or lymphoid tissues (spleen, thymus; axillary, inguinal, and mesenteric lymph nodes) of this animal at the time of euthanasia. Animal 30162 remained healthy and virus negative at 28 months of age; no virus could be detected in an axillary lymph node biopsy specimen taken from this animal at 25 months of age, and the animal had no detectable lymphocyte proliferative responses to SIV antigens (stimulation index, <2).

	DISCUSSION

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The results of the present study provide further insights into the pathogenesis of oral infection of newborn macaques with an SIV isolate, SIVmac055, that has fivefold-reduced in vitro susceptibility to PMPA. In addition, this study addressed how this fivefold-reduced in vitro susceptibility of a virus inoculum to PMPA affects the prophylactic and therapeutic efficacy of early short-term PMPA administration.

In this study, groups of newborn macaques were inoculated orally with SIVmac055. The untreated newborn macaques developed persistently high viremia within 1 to 2 weeks after oral SIVmac055 inoculation. This high viremia during the initial weeks of infection resulted in a rapid and early dissemination of virus to all lymphoid tissues. The inability of the untreated animals to mount strong or persistent anti-SIV IgG responses also indicates virus-induced immunosuppression during these initial weeks of infection (32). This inability to control virus replication resulted in rapidly progressive fatal immunodeficiency within 2 to 3 months. The fulminant disease course observed in the three untreated SIVmac055-infected newborn macaques is similar to that observed previously in newborn macaques following intravenous inoculation with SIVmac055 or following oral or intravenous inoculation with the wild-type parental virus, SIVmac251 (42, 43, 45, 47, 49).

The dose of the SIVmac055 inoculum required to obtain 100% infection of the untreated control animals consisted of two oral inoculations of approximately 15,000 TCID₅₀ each; this dose is in the same range as the dose of 10⁴ to 10⁵ TCID₅₀ that is generally needed to obtain persistent infection with the parenteral wild-type virus, SIVmac251, following mucosal (oral or intravaginal) inoculation (29, 41, 42, 47). This suggests that reduced in vitro drug susceptibility per se does not prohibit a virulent isolate from being transmitted efficiently across mucosal barriers. These findings are consistent with the growing number of reports describing the transmission of HIV-1 variants resistant to widely used anti-HIV drugs and with the observations that the primary course of infection (including peak virus levels) with drug-resistant HIV-1 isolates appears indistinguishable from infection with wild-type HIV (1, 5, 7, 11, 13, 15, 17, 19, 20, 37; P. Hermans, S. Sprecher, and N. Clumeck, Letter, N. Engl. J. Med. 329:1123, 1993; Masquelier et al., Letter).

The study described here also investigated how a reduced in vitro drug susceptibility of the viral inoculum affected the efficacy of PMPA chemoprophylaxis for newborn macaques. Due to pharmacologic and physiologic considerations (including pharmacokinetics, cellular physiology, etc.), it is difficult to extrapolate directly from in vitro drug susceptibility data to chemoprophylactic efficacy in vivo. Accordingly, the use of an animal model is a more reliable way to gain further insights in the clinical implications of reduced in vitro susceptibility. The key questions we addressed in this study were (i) whether a fivefold-reduced in vitro susceptibility to PMPA decreases the efficacy of PMPA to prevent infection of newborn macaques; (ii) whether, if infection is not prevented, this short-term PMPA administration alters the course of infection; and (iii) how SIVmac055 evolves over time in the presence or absence of PMPA treatment.

We demonstrated that PMPA administration for 4 weeks, starting 1 day prior to oral SIVmac055 inoculation, was still effective in protecting two of five newborn macaques against oral SIVmac055 infection. In the animals which became infected despite PMPA administration, virus could already be detected in peripheral blood while these animals were still receiving PMPA administration; this suggests that a longer PMPA treatment regimen, beyond 4 weeks, would not have been more effective in preventing infection in a higher portion of animals. Based on previous PMPA prophylaxis studies by our and other groups, a 4-week PMPA treatment regimen starting prior to virus inoculation is expected to be nearly 100% effective in protecting newborn macaques against infection with wild-type SIVmac251 (39-41, 47). Thus, a fivefold-reduced in vitro susceptibility appears to translate into a partially reduced success of a chemoprophylactic PMPA regimen. This observation suggests that higher levels of drug resistance (such as the >100-fold level of resistance which is frequently seen with ZDV or lamivudine [3TC]) could result in more pronounced failure of chemoprophylaxis with these other antiviral drugs. However, the demonstration that a short PMPA regimen was still partially effective in preventing infection with low-level-PMPA-resistant SIV is still promising and also has important implications regarding the use of PMPA to interrupt HIV transmission from HIV-infected mothers to their infants.

Data from phase I/II human trials have demonstrated that intravenous or oral PMPA treatment of HIV-infected adults resulted in a strong reduction of viral RNA levels, and no emergence of PMPA-resistant HIV mutants was observed during treatment for up to 4 weeks (9; S. G. Deeks, P. Barditch-Crovo, P. S. Lietman, A. Collier, S. Safrin, R. Coleman, K. C. Cundy, and J. O. Kahn, Abstr. 5th Conf. Retroviruses Opportunistic Infect., abstr. 772, 1998; M. Miller, J. M. Cherrington, P. D. Lany, A. S. Mulato, and K. E. Antron, Abstr. 12th World AIDS Conf., abstr. 41218, 1998). These results suggest that it is unlikely that PMPA-resistant HIV-1 mutants would be a problem in clinical settings where PMPA is given for a short period to pregnant women near delivery to reduce intrapartum HIV transmission. In clinical settings where prolonged PMPA treatment is given to HIV-infected pregnant women, K65R HIV-1 mutants with fivefold-reduced susceptibility to PMPA could eventually emerge; our results with SIVmac055, however, suggest that even if the emergence of these mutants leads to a rebound in maternal viremia, PMPA administration could still be continued and could still reduce perinatal HIV transmission. Addition of other antiviral drugs to the PMPA regimen, however, may be advisable in the presence of PMPA-resistant virus, to achieve a maximum level of protection of the newborns.

In this study, no loss of the K65R and other substitutions in RT (N69T, R82K, A158S, and S211N) present in the SIVmac055 inoculum was observed for virus isolates obtained from any of the SIVmac055-infected animals at the time of euthanasia. In addition, no phenotypic reversion to wild-type susceptibility was detected during the observation period. The untreated SIVmac055-infected control animals developed fatal immunodeficiency at 6 to 12 weeks of age, while the SIVmac055-infected animals which received PMPA during the first 4 weeks of infection were euthanized 16 to 59 weeks after PMPA treatment was discontinued. These results indicate that in the absence of PMPA treatment, there was no obvious selection pressure for SIVmac055 to revert to the parental wild-type SIVmac251. Taken together, these results suggest that the combination of the K65R mutation (which confers fivefold-reduced in vitro susceptibility to PMPA [43]) with the four other RT substitutions (N69T, R82K, A158S, and S211N) in SIVmac055 is relatively stable. A number of additional substitutions in RT were, however, detected in virus isolates obtained from the SIVmac055-infected animals; most of these substitutions (K64R and V148G) had already been observed in newborn macaques following intravenous inoculation with SIVmac055 or SIVmac385 (44). Because these substitutions did not further reduce the susceptibility of the virus to PMPA, they may reflect compensatory mutations which emerged as a result of selection pressure toward increased replicative fitness. Taken together, these findings demonstrate that SIVmac055, albeit already quite fit, continues to evolve in vivo.

Our study also demonstrated that for the newborn macaques that became infected with SIVmac055 despite receiving PMPA, the 4 weeks of PMPA treatment had a strong beneficial effect, since their disease-free survival was significantly prolonged. In a previous study, short-term (14 to 60 days) PMPA treatment starting 5 days after infection of newborn macaques with wild-type, drug-susceptible SIVmac251 resulted in a dramatic suppression of virus levels during PMPA treatment. Even though virus replication increased in most animals once PMPA treatment was withdrawn, this early short-term PMPA administration had a pronounced effect on reducing systemic virus dissemination, augmenting antiviral immune responses, and delaying the disease course (45). These data suggested that very early events during viral infection determine the ultimate disease course in simian AIDS. In the present study, we confirmed and extended this observation by using a virus isolate with partial resistance to PMPA and by showing that even a relatively minor delay or suppression of the initial viremia favorably altered the subsequent disease course. Accordingly, the results of this study provide further strong support for the use of potent anti-HIV drug treatment for human newborns and, by extension to adults, immediately following exposure to HIV. Any intervention that suppresses viremia during the initial weeks of infection is likely to produce long-term clinical benefits (21, 23, 34, 35).