Ross P. Tarara

Passive Immunization of Newborn Rhesus Macaques Prevents Oral Simian Immunodeficiency Virus Infection

To determine if passively acquired antiviral antibodies modulate virus transmission and disease progression in human pediatric AIDS, the potential of pre- and postexposure passive immunization with hyperimmune serum to prevent oral simian immunodeficiency virus (SIV) infection or disease progression in newborn rhesus macaques was tested. Untreated neonates became infected after oral SIV inoculation and had high viremia, and most animals developed fatal AIDS within 3 months. In contrast, SIV hyperimmune serum given subcutaneously prior to oral SIV inoculation protected 6 newborns against infection. When this SIV hyperimmune serum was given to 3 newborns 3 weeks after oral SIV inoculation, viremia was not reduced, and all 3 infants died within 3 months of age due to AIDS and immune-complex disease. These results suggest that passively acquired antihuman immunodeficiency virus (HIV) IgG may decrease perinatal HIV transmission. However, anti-HIV IgG may not impart therapeutic benefit to infants with established HIV infection.

Biological Effects of Short-Term or Prolonged Administration of 9-[2-(Phosphonomethoxy)Propyl]Adenine (Tenofovir) to Newborn and Infant Rhesus Macaques

ABSTRACT The reverse transcriptase inhibitor 9-[2-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir) was previously found to offer strong prophylactic and therapeutic benefits in an infant macaque model of pediatric human immunodeficiency virus (HIV) infection. We now summarize the toxicity and safety of PMPA in these studies. When a range of PMPA doses (4 to 30 mg/kg of body weight administered subcutaneously once daily) was administered to 39 infant macaques for a short period of time (range, 1 day to 12 weeks), no adverse effects on their health or growth were observed; this included a subset of 12 animals which were monitored for more than 2 years. In contrast, daily administration of a high dose of PMPA (30 mg/kg subcutaneously) for prolonged periods of time (>8 to 21 months) to 13 animals resulted in a Fanconi-like syndrome (proximal renal tubular disorder) with glucosuria, aminoaciduria, hypophosphatemia, growth restriction, bone pathology (osteomalacia), and reduced clearance of PMPA. The adverse effects were reversible or were alleviated following either complete withdrawal of PMPA treatment or reduction of the daily regimen from 30 mg/kg to 2.5 to 10 mg/kg subcutaneously. Finally, to evaluate the safety of a prolonged low-dose treatment regimen, two newborn macaques were started on a 10-mg/kg/day subcutaneous regimen; these animals are healthy and have normal bone density and growth after 5 years of daily treatment. In conclusion, our findings suggest that chronic daily administration of a high dose of PMPA results in adverse effects on kidney and bone, while short-term administration of relatively high doses and prolonged low-dose administration are safe.

Social Stress Enhances Sympathetic Innervation of Primate Lymph Nodes: Mechanisms and Implications for Viral Pathogenesis

Behavioral processes regulate immune system function in part via direct sympathetic innervation of lymphoid organs, but little is known about the factors that regulate the architecture of neural fibers in lymphoid tissues. In the present study, we find that experimentally imposed social stress can enhance the density of catecholaminergic neural fibers within axillary lymph nodes from adult rhesus macaques. This effect is linked to increased transcription of the key sympathetic neurotrophin nerve growth factor and occurs predominately in extrafollicular regions of the paracortex that contain T-lymphocytes and macrophages. Functional consequences of stress-induced increases in innervation density include reduced type I interferon response to viral infection and increased replication of the simian immunodeficiency virus. These data reveal a surprising degree of behaviorally induced plasticity in the structure of lymphoid innervation and define a novel pathway by which social factors can modulate immune response and viral pathogenesis.

Immunization of Newborn Rhesus Macaques with Simian Immunodeficiency Virus (SIV) Vaccines Prolongs Survival after Oral Challenge with Virulent SIVmac251

ABSTRACT There is an urgent need for active immunization strategies that, if administered shortly after birth, could protect infants in developing countries from acquiring human immunodeficiency virus (HIV) infection through breast-feeding. Better knowledge of the immunogenic properties of vaccine candidates in infants and of the effect of maternal antibodies on vaccine efficacy will aid in the development of such a neonatal HIV vaccine. Simian immunodeficiency virus (SIV) infection of infant macaques is a useful animal model of pediatric HIV infection with which to address these questions. Groups of infant macaques were immunized at birth and 3 weeks of age with either modified vaccinia virus Ankara (MVA) expressing SIV Gag, Pol, and Env (MVA-SIVgpe) or live-attenuated SIVmac1A11. One MVA-SIVgpe-immunized group had maternally derived anti-SIV antibodies prior to immunization. Animals were challenged orally at 4 weeks of age with a genetically heterogeneous stock of virulent SIVmac251. Although all animals became infected, the immunized animals mounted better antiviral antibody responses, controlled virus levels more effectively, and had a longer disease-free survival than the unvaccinated infected monkeys. Maternal antibodies did not significantly reduce the efficacy of the MVA-SIVgpe vaccine. In conclusion, although the tested vaccines delayed the onset of AIDS, further studies are warranted to determine whether a vaccine that elicits stronger early immune responses at the time of virus exposure may be able to prevent viral infection or AIDS in infants.

Prophylactic and Therapeutic Benefits of Short-Term 9-[2-(R)-(Phosphonomethoxy)Propyl]Adenine (PMPA) Administration to Newborn Macaques following Oral Inoculation with Simian Immunodeficiency Virus with Reduced Susceptibility to PMPA

ABSTRACT Simian immunodeficiency virus (SIV) infection of newborn macaques is a useful animal model of human pediatric AIDS to study pathogenesis and to develop intervention strategies aimed at preventing infection or delaying disease progression. In previous studies, we demonstrated that 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir) was highly effective in protecting newborn macaques against infection with virulent wild-type (i.e., drug-susceptible) SIVmac251. In the present study, we determined how reduced drug susceptibility of the virus inoculum affects the chemoprophylactic success. SIVmac055 is a virulent isolate that has a fivefold-reduced in vitro susceptibility to PMPA, associated with a K65R mutation and additional amino acid changes (N69T, R82K, A158S, S211N) in reverse transcriptase (RT). Eight newborn macaques were inoculated orally with SIVmac055. The three untreated control animals became SIVmac055 infected; these animals had persistently high viremia and developed fatal immunodeficiency within 3 months. Five animals were treated once daily with PMPA (at 30 mg/kg of body weight) for 4 weeks, starting 24 h prior to oral SIVmac055 inoculation. Two of the five PMPA-treated animals had no evidence of infection. The other three PMPA-treated infant macaques became infected but had a delayed viremia, enhanced antiviral antibody responses, and a slower disease course (AIDS in 5 to 15 months). No reversion to wild-type susceptibility or loss of the K65R mutation was detected in virus isolates from any of the PMPA-treated or untreated SIVmac055-infected animals. Several additional amino acid changes developed in RT, but they were not exclusively associated with PMPA therapy. The results of this study suggest that prophylactic administration of PMPA to human newborns and to adults following exposure to human immunodeficiency virus will still be beneficial even in the presence of viral variants with reduced susceptibility to PMPA.

Experimental Coinfection of Rhesus Macaques with Rhesus Cytomegalovirus and Simian Immunodeficiency Virus: Pathogenesis

ABSTRACT Human cytomegalovirus (HCMV) possesses low pathogenic potential in an immunocompetent host. In the immunosuppressed host, however, a wide spectrum of infection outcomes, ranging from asymptomatic to life threatening, can follow either primary or nonprimary infection. The variability in the manifestations of HCMV infection in immunosuppressed individuals implies that there is a threshold of host antiviral immunity that can effectively limit disease potential. We used a nonhuman primate model of CMV infection to assess the relationship between CMV disease and the levels of developing anti-CMV immunity. Naive rhesus macaques were inoculated with rhesus cytomegalovirus (RhCMV) followed 2 or 11 weeks later by inoculation with pathogenic simian immunodeficiency virus SIVmac239. Two of four monkeys inoculated with SIV at 2 weeks after inoculation with RhCMV died within 11 weeks with simian AIDS (SAIDS), including activated RhCMV infection. Neither animal had detectable anti-SIV antibodies. The other two animals died 17 and 27 weeks after SIV inoculation with either SAIDS or early lymphoid depletion, although no histological evidence of activated RhCMV was observed. Both had weak anti-SIV antibody titers. RhCMV antibody responses for this group of monkeys were significantly below those of control animals inoculated with only RhCMV. In addition, all animals of this group had persistent RhCMV DNA in plasma and high copy numbers of RhCMV in tissues. In contrast, animals that were inoculated with SIV at 11 weeks after RhCMV infection rarely exhibited RhCMV DNA in plasma, had low copy numbers of RhCMV DNA in most tissues, and did not develop early onset of SAIDS or activated RhCMV. SIV antibody titers were mostly robust and sustained in these monkeys. SIV inoculation blunted further development of RhCMV humoral responses, unlike the normal pattern of development in control monkeys following RhCMV inoculation. Anti-RhCMV immunoglobulin G levels and avidity were slightly below control values, but levels maintained were higher than those observed following SIV infection at 2 weeks after RhCMV inoculation. These findings demonstrate that SIV produces long-lasting insults to the humoral immune system beginning very early after SIV infection. The results also indicate that anti-RhCMV immune development at 11 weeks after infection was sufficient to protect the host from acute RhCMV sequelae following SIV infection, in contrast to the lack of protection afforded by only 2 weeks of immune response to RhCMV. As previously observed, monkeys that were not able to mount a significant immune response to SIV were the most susceptible to SAIDS, including activated RhCMV infection. Rapid development of SAIDS in animals inoculated with SIV 2 weeks after RhCMV inoculation suggests that RhCMV can augment SIV pathogenesis, particularly during primary infection by both viruses.

Virulence and Reduced Fitness of Simian Immunodeficiency Virus with the M184V Mutation in Reverse Transcriptase

ABSTRACT Drug-resistant mutants with a methionine-to-valine substitution at position 184 of reverse transcriptase (M184V) emerged within 5 weeks of initiation of therapy in four newborn macaques infected with simian immunodeficiency virus (SIVmac251) and treated with lamivudine (3TC) or emtricitabine [(−)-FTC] (two animals per drug). Thus, this animal model mimics the rapid emergence of M184V mutants of HIV-1 during 3TC therapy of human patients. One animal of each treatment group developed fatal immunodeficiency at 12 weeks of age, which is similar to the rapid disease course seen in most untreated SIVmac251-infected infant macaques. To further evaluate the effect of the M184V mutation on viral fitness and virulence, groups of juvenile macaques were inoculated with the molecular clone SIVmac239 with either the wild-type sequence (group A [n = 5]) or the M184V sequence (SIVmac239-184V; group B [n = 5] and group C [n = 2]). The two SIVmac239-184V-infected animals of group C did not receive any drug treatment, and in both animals the virus population reverted to predominantly wild type (184M) by 8 weeks after inoculation. The other five SIVmac239-184V-infected animals (group B) were treated with (−)-FTC to prevent reversion. Although virus levels 1 week after inoculation were lower in the SIVmac239-184V-infected macaques than in the SIVmac239-infected animals, no significant differences were observed from week 2 onwards. Two animals in each group developed AIDS and were euthanized, while all other animals were clinically stable at 46 weeks of infection. These data demonstrate that the M184V mutation in SIV conferred a slightly reduced fitness but did not affect disease outcome.

The clinical benefits of tenofovir for simian immunodeficiency virus-infected macaques are larger than predicted by its effects on standard viral and immunologic parameters.

Summary:Previous studies have demonstrated that tenofovir (9-[2-(phosphonomethoxy)propyl]adenine; PMPA) treatment is usually very effective in suppressing viremia in macaques infected with simian immunodeficiency virus (SIV). The present study focuses on a subset of infant macaques that were chronically infected with highly virulent SIVmac251, and for which prolonged tenofovir treatment failed to significantly suppress viral RNA levels in plasma despite the presence of tenofovirsusceptible virus at the onset of therapy. While untreated animals with similarly high viremia developed fatal immunodeficiency within 3–6 months, these tenofovir-treated animals had significantly improved survival (up to 3.5 years). This clinical benefit occurred even in animals for which tenofovir had little or no effect on CD4+ and CD8+ lymphocyte counts and antibody responses to SIV and test antigens. Thus, the clinical benefits of tenofovir were larger than predicted by plasma viral RNA levels and other routine laboratory parameters.

Recombinant Bacille Calmette-Guérin Expressing the Measles Virus Nucleoprotein Protects Infant Rhesus Macaques from Measles Virus Pneumonia

Measles virus infection continues to be a major cause of infant mortality. There is a need for a measles vaccine that can be administered at birth in the presence of maternal neutralizing antibody. Infant rhesus monkeys were immunized with recombinant bacille Calmette-Guérin expressing the full-length measles virus nucleoprotein (BCG-N) and subsequently challenged with measles virus. Nucleoprotein-specific lymphocyte proliferative responses were detected in the absence of anti-N antibody after vaccination. Vaccination with BCG-N did not prevent systemic measles virus infection; however, there was a significant reduction of lung inflammation after challenge. Virus titers in lymph nodes were significantly lower, and the duration of nasopharyngeal viral shedding was shorter in some vaccinated monkeys after challenge. These results suggest that measles virus-specific T cells were primed by BCG-N vaccination and that they prevented virus-induced lung pathology.

Enhanced Replication of Simian Immunodeficiency Virus Adjacent to Catecholaminergic Varicosities in Primate Lymph Nodes

ABSTRACT Clinical and in vitro studies have shown that activity of the autonomic nervous system (ANS) can stimulate lentivirus replication. To define the potential anatomical basis for this effect, we analyzed the spatial relationship between catecholaminergic neural fibers and sites of simian immunodeficiency virus (SIV) replication in lymph nodes from rhesus macaques experimentally infected with SIVmac251. Viral replication was mapped by in situ hybridization for SIV env, gag, and nef RNA, and catecholaminergic varicosities from the ANS were mapped by sucrose phosphate glyoxylic acid chemofluorescence. Spatial statistical analyses showed that the likelihood of active SIV replication increased by 3.9-fold in the vicinity of catecholaminergic varicosities (P < 0.0001). The densities of both ANS innervation and SIV replication differed across cortical, paracortical, and medullary regions of the lymph node, but analyses of each region separately continued to show increased replication of SIV adjacent to catecholaminergic varicosities. Ancillary analyses ruled out the possibility that SIV-induced alterations in lymph node architecture might create a spurious spatial association. These data support human clinical studies and in vitro molecular analyses showing that catecholamine neurotransmitters from the ANS can increase lentiviral replication by identifying a specific anatomic context for interactions between ANS neural fibers and replication of SIV in lymphoid tissue.