Bruce J. Bernacky

Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection.

Although maternal human immunodeficiency virus type 1 (HIV-1) transmission occurs during gestation, intrapartum and postpartum (by breast-feeding), 50–70% of all infected children seem to acquire HIV-1 shortly before or during delivery. Epidemiological evidence indicates that mucosal exposure is an important aspect of intrapartum HIV transmission. A simian immunodeficiency virus (SIV) macaque model has been developed that mimics the mucosal exposure that can occur during intrapartum HIV-1 transmission. To develop immunoprophylaxis against intrapartum HIV-1 transmission, we used SHIV–vpu+ (refs. 5,6), a chimeric simian–human virus that encodes the env gene of HIV-IIIB. Several combinations of human monoclonal antibodies against HIV-1 have been identified that neutralize SHIV–vpu+ completely in vitro through synergistic interaction. Here, we treated four pregnant macaques with a triple combination of the human IgG1 monoclonal antibodies F105, 2G12 and 2F5. All four macaques were protected against intravenous SHIV–vpu+ challenge after delivery. The infants received monoclonal antibodies after birth and were challenged orally with SHIV–vpu+ shortly thereafter. We found no evidence of infection in any infant during 6 months of follow-up. This demonstrates that IgG1 monoclonal antibodies protect against mucosal lentivirus challenge in neonates. We conclude that epitopes recognized by the three monoclonal antibodies are important determinants for achieving substantial protection, thus providing a rational basis for AIDS vaccine development.

Postnatal Passive Immunization of Neonatal Macaques with a Triple Combination of Human Monoclonal Antibodies against Oral Simian-Human Immunodeficiency Virus Challenge

ABSTRACT To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351–357, 1994). Using this model, neonates were protected from mucosal SHIV-vpu+challenge by pre- and postnatal treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and 2F5 (Baba et al., Nat. Med. 6:200–206, 2000). In the present study, we used this MAb combination only postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu+ challenge, while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination against the more pathogenic SHIV89.6P, a chimeric virus encodingenv of the primary HIV89.6. Remarkably, the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4+ T-cell decline. In contrast, all control animals had dramatic drops in their CD4+ T cells by 2 weeks postexposure. We conclude that our triple MAb combination partially protected against mucosal challenge with the highly pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1.

Quantification of thymic function by measuring T cell receptor excision circles within peripheral blood and lymphoid tissues in monkeys.

The thymus is the primary organ responsible for the production of mature TCR α / β T cells. Quantification of a DNA excision circle that is produced during TCR rearrangement, termed a signal joint TCR rearrangement excision circle (sjTREC) can be used as a measure of thymic function. Here sjTREC measurement has been applied to two monkey species used as animal models of human disease, rhesus macaques (Asian origin) and sooty mangabeys (African origin). Initial PCR analysis determined that the TCR δRec‐ΨJα rearrangement leading to sjTREC formation occurs in both species. Primers to a DNA sequence conserved in macaques, mangabeys and humans were used in a quantitative competitive PCR assay to quantify sjTREC. We found that as in humans, sjTREC in these two monkey species decline with age. sjTREC are first generated in thymocytes during the early stages of TCR rearrangement. Lymph node CD4+ and CD8+ T cells contain more sjTREC than peripheral blood T cell populations, suggesting that recent thymic emigrants home to the lymphoid tissues. The sjTREC level is significantly higher within the peripheral blood CD4+ and CD8+ T cells of mangabeys compared to macaques. Removal of the thymus in four macaques led to a profound decrease in peripheral blood sjTREC level by 1 year post‐thymectomy, indicating the lack of a significant extra‐thymic source of peripheral naive T cells in macaques. Our results indicate that production, trafficking, and proliferation of recent thymic emigrants in these two monkey species represents a useful animal model system for understanding human immunological disorders.

A Peptidomimetic Targeting White Fat Causes Weight Loss and Improved Insulin Resistance in Obese Monkeys

A peptide-based drug that targets the vasculature of adipose tissue induces weight loss and improves metabolic function in spontaneously obese monkeys. Fat Monkeys Get Trim The rapidly increasing rate of obesity worldwide is one of the biggest health challenges facing society today. Unlike related threats such as cancer, cardiovascular disease, and diabetes, very few approved drugs are available to treat obesity despite some promising early-stage candidates. In a new study, Barnhart and colleagues take a fresh approach to treating obesity by developing a peptide-like molecule that targets the blood vessels that feed fat tissue. They test their peptidomimetic called adipotide in obese monkeys and show that it both reduces fat tissue and decreases resistance to insulin. Adipotide is a short peptide-based agent that selectively targets a receptor expressed by the vascular endothelial cells that comprise the blood vessels that support subcutaneous and visceral fat. This peptidomimetic carries a molecule that, once internalized by the endothelial cells, causes them to undergo programmed cell death, thereby inducing gradual elimination of excess fat. In placebo-controlled experiments, spontaneously obese rhesus monkeys treated with adipotide for 28 days showed a 7 to 15% weight loss as well as improved insulin resistance. Two forms of imaging revealed that the weight loss occurred primarily through a reduction in fat tissue and did not reflect fluid loss or muscle wasting. Monkeys treated with adipotide displayed a 38% reduction in total body fat and a 27% reduction in abdominal fat compared to pretreatment baseline values. Early weight loss drug candidates are typically screened in rodent models of obesity. However, the central nervous system control and metabolic regulation of food intake and fat storage in rodents is quite different from that of monkeys and humans. Spontaneously obese monkeys are a more accurate model of obesity in humans and provide a valuable setting for testing anti-obesity drug candidates. Adipotide therapy resulted in a reduction in body mass, an improvement in insulin resistance, and a decrease in abdominal circumference, key predictors of diabetes in humans. These encouraging results support the further development of adipotide as a potential new prototype drug to combat obesity in humans. Obesity, defined as body mass index greater than 30, is a leading cause of morbidity and mortality and a financial burden worldwide. Despite significant efforts in the past decade, very few drugs have been successfully developed for the treatment of obese patients. Biological differences between rodents and primates are a major hurdle for translation of anti-obesity strategies either discovered or developed in rodents into effective human therapeutics. Here, we evaluate the ligand-directed peptidomimetic CKGGRAKDC-GG-D(KLAKLAK)2 (henceforth termed adipotide) in obese Old World monkeys. Treatment with adipotide induced targeted apoptosis within blood vessels of white adipose tissue and resulted in rapid weight loss and improved insulin resistance in obese monkeys. Magnetic resonance imaging and dual-energy x-ray absorptiometry confirmed a marked reduction in white adipose tissue. At experimentally determined optimal doses, monkeys from three different species displayed predictable and reversible changes in renal proximal tubule function. Together, these data in primates establish adipotide as a prototype in a new class of candidate drugs that may be useful for treating obesity in humans.

Protection of neonatal macaques against experimental SHIV infection by human neutralizing monoclonal antibodies

Neonatal macaques were completely protected against oral challenge with SHIV-vpu+, a simian-human immunodeficiency virus that encodes the envelope gene of a laboratory-adapted HIV strain, by pre- and post-natal treatment with a triple combination of human neutralizing monoclonal antibodies (mAbs). The mAbs were directed either against the CD4 binding site, a glycosylation-dependent gp120 epitope, or against a linear epitope on gp41. This triple combination was highly synergistic in vitro and neutralized primary HIV completely. Subsequently, oral challenge was performed with pathogenic SHIV89.6P, an animal-passaged variant of a chimeric virus that encodes the envelope gene of the primary, dual-tropic HIV89.6. Only post-natal treatment with a similar triple mAb combination was used. One out of 4 mAb-treated infants was completely protected from infection. In the other 3 treated animals, there was a tendency towards lower peak viral RNA loads compared with untreated controls. Two out of 4 mAb-treated infants maintained normal CD4+ T-cell numbers, in contrast to all controls that had steep declines at 2 weeks post-challenge. We conclude that the triple mAb combination significantly protected the neonates, even against mucosal challenge with pathogenic SHIV89.6P. Passively administered synergistic human mAbs may play a role in preventing mother-infant transmission of HIV, both against intrapartum transmission as well as against infection through breast milk. As passive immunization is a tool to assess correlates of immune protection, we conclude that the epitopes recognized by the mAbs in our combinations are important for AIDS vaccine development. Future passive immunization studies may reveal other important conserved epitopes.

Postnatal pre- and postexposure passive immunization strategies: protection of neonatal macaques against oral simian–human immunodeficiency virus challenge

Simian–human immunodeficiency viruses (SHIV) allow the evaluation of antiviral strategies that target the envelope glycoproteins of the human immunodeficiency virus 1 (HIV‐1) in macaques. We previously protected neonates from oral challenge with cell‐free SHIV‐vpu+ by passive immunization with synergistic human neutralizing monoclonal antibodies (mAbs) (Baba et al., Nat Med 6:200–206, 2000). mAbs were administered prenatally to pregnant dams and postnatally to the neonates. Here, we used solely postnatal or postexposure mAb treatment, thus significantly reducing the amount of mAbs necessary. All neonatal monkeys were also protected with these abbreviated mAb regimens. Our results are directly relevant for humans because we used mAbs that target HIV‐1 envelope glycoproteins. Thus, the large‐scale use of passive immunization with neutralizing mAbs may be feasible in human neonates. The mAbs, being natural human proteins, can be expected to have low toxicity. Passive immunization has promise to prevent intrapartum as well as milk‐borne virus transmission from HIV‐1‐infected women to their infants.

Protection against chronic infection and AIDS by an HIV envelope peptide-cocktail vaccine in a pathogenic SHIV-rhesus model

Based on our prior studies in mouse, monkey, chimpanzee, and human experimental systems, we identified six peptides encoded by highly conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope gene that selectively induce cellular immune responses in the absence of anti-viral antibody production. We tested a cocktail of the six peptides as a prototype vaccine for protection from simian human immunodeficiency virus (SHIV) infection and acquired immunodeficiency syndrome (AIDS) in a rhesus monkey model. Three monkeys were vaccinated with the peptide cocktail in Freunds adjuvant followed by autologous dendritic cells (DC) pulsed with these peptides. All the vaccinated animals exhibited significant induction of T-cell proliferation and cytotoxic T lymphocytes (CTL) responses, but no neutralizing antibodies. Two control mock-vaccinated monkeys showed no specific immune responses. Upon challenge with the pathogenic SHIV(KU-2), both the control and vaccinated monkeys were infected, but efficient clearance of virus-infected cells was observed in all the three vaccinated animals within 14 weeks. These animals also experienced a boosting of antiviral cellular immune responses after infection, and maintained antigen-specific IFN-gamma-producing cells in circulation beyond 42 weeks post-challenge. In contrast, the two mock-vaccinated monkeys had low to undetectable cellular immune responses and maintained significant levels of viral-infected cells and infectious virus in circulation. Further, in both the control monkeys plasma viremia was detectable beyond 38 weeks post-challenge indicating chronic phase infection. In one control monkey, the CD4+ cells dropped to very low levels by 2 weeks post-challenge and became undetectable by week 39 coinciding with high plasma viremia and AIDS, which included cachexia and ataxia. These results serve as proof of principle for the effectiveness of the HIV envelope peptide cocktail vaccine against chronic infection and AIDS, and support the development of multivalent peptide-based vaccine as a viable strategy to induce cell-mediated immunity (CMI) for protection against HIV and AIDS in humans.

Oral Transmission of Primate Lentiviruses

Oral transmission of human immunodeficiency virus type 1 (HIV-1) is well documented in children who become infected postnatally through breast milk. In contrast, epidemiologic surveys have yielded conflicting data regarding oral HIV-1 transmission among adults, even though case reports have described seroconversion and the development of AIDS in adults whose only risk was oral-genital contact. To study oral virus transmission in primate models, we exposed rhesus macaques of various ages to cell-free simian immunodeficiency virus (SIV), including uncloned and molecularly cloned viruses. In neonates, viremia and AIDS developed after nontraumatic oral exposure to several SIV strains. Furthermore, chimeric simian human immunodeficiency viruses containing the HIV-1 envelope can also cross intact upper gastrointestinal mucosal surfaces in neonates. In adult macaques, infection and AIDS have resulted from well-controlled, nontraumatic, experimental oral exposure to different strains of SIV. These findings have implications for the risks of HIV-1 transmission during oral-genital contact.

Passive immunization against oral AIDS virus transmission: An approach to prevent mother‐to‐infant HIV‐1 transmission?

To develop immunoprophylaxis regimens against mother‐to‐child human immunodeficiency virus type 1 (HIV‐1) transmission, we established a simian–human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al.: AIDS Res Hum Retroviruses 10:351–357, 1994). We protected four neonates from oral SHIV‐vpu+ challenge by ante‐ and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti‐HIV‐1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann‐Lehmann et al.: Nature Med 6:200–206, 2000), which recognize the CD4‐binding site of Env, a glycosylation‐dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV‐vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb‐treated neonates was protected from infection and two maintained normal CD4+ T‐cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV‐1 transmission in humans.

Spontaneous anaplastic large cell lymphoma in a chimpanzee: A clinicopathological and immunohistochemical study

Abstract: An anaplastic large cell lymphoma with disseminated abdominal metastases was diagnosed in a 35‐year‐old male chimpanzee. Clinically, the animal displayed lethargy, weight loss, ascites, and abdominal distention. Imaging studies showed several large abdominal masses. At necropsy, variably sized masses of neoplastic mesenteric lymph nodes that encompassed several intestinal loops were present throughout the abdomen. The largest mass measured 9 × 5 cm and had cauliflower‐like protrusions into the jejunal lumen. The entire abdominal cavity was covered by a sheet of neoplastic tissue. Histopathologically, the tumor consisted of solid sheets of proliferating lymphoid cells forming a cohesive growth that filled the lymph node sinuses. The tumor had invaded the intestinal wall. Anaplastic large cell lymphoma was diagnosed from immunohistochemistry findings on the basis of positive reaction to the CD3 and CD30 markers and negative reaction to the CD20 marker. Serologic analysis revealed positive titers for Epstein‐Barr, cytomegalo‐, and varicella‐zoster viruses. Both serologic and virologic studies showed no evidence of detectable retroviral infection. This type of tumor has not been reported before in the chimpanzee.