Michael Murphey-Corb

Necropsy Findings in Rhesus Monkeys Experimentally Infected with Cultured Simian Immunodeficiency Virus (SIV)/Delta

Lesions induced in rhesus monkeys by different isolates of simian immunodeficiency virus (SIV)/Delta were studied at necropsy. Four groups of monkeys were inoculated with SIV/Delta isolated from other experimentally infected rhesus monkeys, while one group was inoculated with SIV/Delta from an asymptomatic mangabey monkey. Three rhesus isolates and the mangabey isolate were virulent, killing 75–100% of infected monkeys. One rhesus isolate, which had been extensively passaged in vitro, was attenuated but was restored to virulence by single animal passage. Clinically, infected monkeys had lymphadenopathy, splenomegaly, diarrhea, and a rash. Most monkeys died of enteric disease. The following lesions were seen: weight loss, thymic atrophy, lymphoid atrophy, bone marrow hyperplasia, encephalitis, colitis, amyloidosis, hepatitis, glomerulosclerosis, and the presence of syncytial cells. One Rh Epstein-Barr virus (EBV)-related lymphoma occurred. Opportunistic agents were identified: cytomegalovirus, adenovirus, Cryptosporidia, and Pneumocystis. Shigella and Campylobacter often caused colitis.

Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines.

Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1α (MIP-1α), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3β and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.

Infection and AIDS in Adult Macaques After Nontraumatic Oral Exposure to Cell-Free SIV

Unprotected receptive anal intercourse is a well-recognized risk factor for infection with human immunodeficiency virus-type 1 (HIV-1). Isolated human case reports have implicated HIV-1 transmission by oral-genital exposure. Adult macaques exposed nontraumatically to cell-free simian immunodeficiency virus (SIV) through the oral route became infected and developed acquired immunodeficiency syndrome (AIDS). The minimal virus dose needed to achieve systemic infection after oral exposure was 6000 times lower than the minimal dose required to achieve systemic infection after rectal exposure. Thus, unprotected receptive oral intercourse, even in the absence of mucosal lesions, should be added to the list of risk behaviors for HIV-1 transmission.

Induction of Mucosal Protection against Primary, Heterologous Simian Immunodeficiency Virus by a DNA Vaccine

ABSTRACT An effective vaccine against human immunodeficiency virus (HIV) should protect against mucosal transmission of genetically divergent isolates. As a safe alternative to live attenuated vaccines, the immunogenicity and protective efficacy of a DNA vaccine containing simian immunodeficiency virus (SIV) strain 17E-Fr (SIV/17E-Fr) gag-pol-env was analyzed in rhesus macaques. Significant levels of cytotoxic T lymphocytes (CTL), but low to undetectable serum antibody responses, were observed following multiple immunizations. SIV-specific mucosal antibodies and CTL were also detected in rectal washes and gut-associated lymphoid tissues, respectively. Vaccinated and naive control monkeys were challenged intrarectally with SIV strain DeltaB670 (SIV/DeltaB670), a primary isolate whose env is 15% dissimilar to that of the vaccine strain. Four of seven vaccinees were protected from infection as determined by the inability to identify viral RNA or DNA sequences in the peripheral blood and the absence of anamnestic antibody responses postchallenge. This is the first report of mucosal protection against a primary pathogenic, heterologous isolate of SIV by using a commercially viable vaccine approach. These results support further development of a DNA vaccine for protection against HIV.

Species-specific variation in SIV disease progression between Chinese and Indian subspecies of rhesus macaque

Abstract: The shortage of rhesus macaques of Indian origin for acquired immune deficiency syndrome (AIDS) research has prompted a search for an alternate species. As rhesus macaques of Chinese origin are more readily obtainable, we have defined the parameters of infection in seven members of this subspecies with the primary virulent isolate, SIV/DeltaB670. Viremic peaks and set points as determined by real time polymerase chain reaction were, in general, lower than that observed in Indian origin rhesus macaques. As expected, these values were associated with maintenance of CD4+ lymphocytes and significantly longer survival, with six of seven Chinese origin animals living significantly longer than Indian origin rhesus macaques. Interestingly, these findings were associated with a selective amplification of one of two major phylogenetic groups found within the inoculum. This observation is in contrast to Indian origin animals where both phylogenetic groups are commonly identified. Together, these data suggest prudence in the design of experimental protocols using rhesus macaques of Chinese origin where survival and rapid loss of CD4+ lymphocytes are desired endpoints.

YKL-40, a Marker of Simian Immunodeficiency Virus Encephalitis, Modulates the Biological Activity of Basic Fibroblast Growth Factor

Human immunodeficiency virus encephalitis causes dementia in acquired immune deficiency syndrome patients. Using proteomic analysis of postmortem cerebrospinal fluid (CSF) and brain tissue from the simian immunodeficiency virus primate model, we demonstrate here a specific increase in YKL-40 that was tightly associated with lentiviral encephalitis. Longitudinal analysis of CSF from simian immunodeficiency virus-infected pigtailed macaques showed an increase in YKL-40 concentration 2 to 8 weeks before death from encephalitis. This increase in YKL-40 correlated with an increase in CSF viral load; it may therefore represent a biomarker for the development of encephalitis. Analysis of banked human CSF from human immunodeficiency virus-infected patients also demonstrated a correlation between YKL-40 concentration and CSF viral load. In vitro studies demonstrated increased YKL-40 expression and secretion by macrophages and microglia but not by neurons or astrocytes. We found that YKL40 displaced extracellular matrix-bound basic fibroblast growth factor (bFGF) as well as inhibited the mitogenic activity of both fibroblast growth factor receptor 1-expressing BaF3 cells and bFGF-induced axonal branching in hippocampal cultures. Taken together, these findings demonstrate that during lentiviral encephalitis, YKL-40 may interfere with the biological activity of bFGF and potentially of other heparin-binding growth factors and chemokines that can affect neuronal function or survival.

Gene gun-based nucleic acid immunization alone or in combination with recombinant vaccinia vectors suppresses virus burden in rhesus macaques challenged with a heterologous SIV

Gene gun‐based DNA immunization alone or in combination with recombinant vaccinia vectors was evaluated for the ability to elicit protective immune responses in rhesus macaques challenged with a pathogenic, heterologous simian immunodeficiency virus (SIV). Six monkeys primed with seven consecutive doses of DNA encoding SIV mac239 gpl20 and gpl60 (DNA+DNA) were divided into two groups. Three of these animals received another DNA booster immunization and the remaining three received a booster immunization containing a homologous, live recombinant vaccinia virus expressing SIV mac251gpl60 (DNA+VAC). In addition, a group of 15 animals primed with recombinant vaccinia vectors were divided into two groups. One group of six monkeys received another immunization of vaccinia (VAC+VAC) and the other nine animals received a DNA (mac239) booster immunization (VAC+DNA). Geometric mean end‐point IgG titres in the DNA+VAC and VAC+DNA groups were substantially higher than the responses seen in the DNA+VAC and VAC+DNA groups, demonstrating a synergistic relationship between DNA‐based vaccines and recombinant vaccinia virus‐based vaccines. All vaccinates and five naive controls were challenged 19 weeks after the final booster immunization with 10 animal infectious doses of SIVdelta/b670. The vaccines did not prevent infection. However, all vaccine groups showed significant virus load reductions from seven to 56 days post challenge when compared to controls. Although the DNA+DNA group developed the lowest prechallenge antibody responses, the most significant reduction (200‐fold) in virus load was associated with this group. In addition, a significant delay in CD4+ T cell loss relative to controls was observed in the DNA+DNA group. These results demonstrate that a gene gun‐based DNA vaccine provided some attenuation of infection and CD4+ T cell loss after a heterologous challenge.

Induction of immunodeficiency virus-specific immune responses in rhesus monkeys following gene gun-mediated DNA vaccination

Abstract: The Accell® gene delivery system (gene gun) was used to deliver gold particles coated with HIV‐1LAI and SIVmac239 expression constructs into the epidermis of rhesus macaques, resulting in the elicitation of env‐ and gag‐specific humoral responses. One microgram of vector DNA per dose was sufficient to induce immune responses in monkeys using SIVmac239 gp160 and gp120 vectors driven by the CMV‐intron A promoter. Several parameters, including the identity of the vector, the length of the rest period between immunizations, the number of immunizations, and the amount of DNA per immunization, are all important in designing an optimal DNA immunization regimen. In addition, gene gun‐based DNA immunization using low efficiency expression vectors is an effective means of priming for the induction of vigorous antibody responses in macaques following boosting with recombinant subunits.

The Effect of Chronic Binge Ethanol Consumption on the Primary Stage of SIV Infection in Rhesus Macaques

BACKGROUND Alcohol abuse and infection with HIV individually compromise immune function, but the consequence of both conditions together is poorly understood owing to the difficulties of performing appropriate studies in human subjects. Simian immunodeficiency virus (SIV) infection of rhesus macaques is considered to closely model HIV disease in that the virus infects CD4+ cells and this infection leads to a similar AIDS state. This study was initiated to study the combined effects of chronic binge alcohol consumption on the primary stage of SIV infection. METHODS Rhesus macaques were administered alcohol or isocaloric sucrose via a permanently indwelling intragastric catheter 4 consecutive days per week for the duration of the study. Doses were individualized to achieve plasma alcohol concentrations of 50-60 mM over a 5-hr period. After 3 months, animals were inoculated intravenously with 10,000 times the ID(50) (50% infective dose) of SIV(DeltaB670) at the conclusion of an alcohol session and followed for 2 months postinoculation. RESULTS At 1 week, plasma SIV RNA was greater than 60-fold higher in alcohol-consuming animals compared with sucrose controls. Likewise, alcohol consumption enhanced the SIV-induced increase in cell cycling T lymphocytes (i.e., cells expressing Ki67 protein) in blood. These differences between alcohol- and sucrose-treated animals were not sustained during the observation period. Peak viral load occurred 2 weeks post-SIV inoculation at 7.6 +/- 4.2 and 5.2 +/- 3.1 x 106 copies/ml in alcohol- versus sucrose-consuming animals, respectively. Blood CD4+ lymphocyte numbers were decreased 1 and 2 months after SIV inoculation to a similar degree in both sucrose-control and alcohol-treated animals. CONCLUSIONS The consequence of the early rise in viral load and increase in lymphocyte turnover seen with excess alcohol consumption is unknown. We hypothesize that alcohol intoxication may increase the susceptibility of the host to HIV/SIV infection. This possibility needs to be explored further.

GM-CSF Increases Mucosal and Systemic Immunogenicity of an H1N1 Influenza DNA Vaccine Administered into the Epidermis of Non-Human Primates

Background The recent H5N1 avian and H1N1 swine-origin influenza virus outbreaks reaffirm that the threat of a world-wide influenza pandemic is both real and ever-present. Vaccination is still considered the best strategy for protection against influenza virus infection but a significant challenge is to identify new vaccine approaches that offer accelerated production, broader protection against drifted and shifted strains, and the capacity to elicit anti-viral immune responses in the respiratory tract at the site of viral entry. As a safe alternative to live attenuated vaccines, the mucosal and systemic immunogenicity of an H1N1 influenza (A/New Caledonia/20/99) HA DNA vaccine administered by particle-mediated epidermal delivery (PMED or gene gun) was analyzed in rhesus macaques. Methodology/Principal Findings Macaques were immunized at weeks 0, 8, and 16 using a disposable single-shot particle-mediated delivery device designed for clinical use that delivers plasmid DNA directly into cells of the epidermis. Significant levels of hemagglutination inhibiting (HI) antibodies and cytokine-secreting HA-specific T cells were observed in the periphery of macaques following 1–3 doses of the PMED HA DNA vaccine. In addition, HA DNA vaccination induced detectable levels of HA-specific mucosal antibodies and T cells in the lung and gut-associated lymphoid tissues of vaccinated macaques. Importantly, co-delivery of a DNA encoding the rhesus macaque GM-CSF gene was found to significantly enhance both the systemic and mucosal immunogenicity of the HA DNA vaccine. Conclusions/Significance These results provide strong support for the development of a particle-mediated epidermal DNA vaccine for protection against respiratory pathogens such as influenza and demonstrate, for the first time, the ability of skin-delivered GM-CSF to serve as an effective mucosal adjuvant for vaccine induction of immune responses in the gut and respiratory tract.