Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.     

Persistent infection of chimpanzees with human immunodeficiency virus: serological responses and properties of reisolated viruses.

Persistent infection by human immunodeficiency virus (HIV-1) in the chimpanzee may be valuable for immunopathologic and potential vaccine evaluation. Two HIV strains, the tissue culture-derived human T-cell lymphotropic virus type IIIB (HTLV-IIIB) and in vivo serially passaged lymphadenopathy-associated virus type 1 (LAV-1), were injected intravenously into chimpanzees. Two animals received HTLV-IIIB as either virus-infected H9 cells or cell-free virus. A third animal received chimpanzee-passaged LAV-1. Evaluation of their sera for virus-specific serologic changes, including neutralizations, was done during a 2-year period. During this period all animals had persistently high titers of antibodies to viral core and envelope antigens. All three animals developed a progressively increasing type-specific neutralizing LAV-1 versus HTLV-IIIB antibody titer during the 2-year observation period which broadened in specificity to include HTLV-HIRF, HTLV-IIIMN, and HTLV-IIICC after 6 to 12 months. The antibody titers against both viruses were still increasing by 2 years after experimental virus inoculation. Sera from all animals were capable of neutralizing both homologously and heterologously reisolated virus from chimpanzees. A slightly more rapid type-specific neutralizing response was noted for the animal receiving HTLV-IIIB-infected cells compared with that for cell-free HTLV-IIIB. Sera from all persistently infected chimpanzees were capable of mediating group-specific antibody-mediated complement-dependent cytolysis of HIV-infected cells derived from all isolates tested. Viruses reisolated from all three animals at 20 months after inoculation revealed very similar peptide maps of their respective envelope gp120s, as determined by two-dimensional chymotrypsin oligopeptide analysis. One peptide, however, from the original HTLV-IIIB-inoculated virus was deleted in viruses from all three animals, and in addition, we noted the appearance of a new or modified peptide which was common to LAV-1 as well as to HTLV-IIIB reisolated from infected chimpanzees. It thus appears that a group-specific neutralizing antibody response as well as a group-specific cytotoxic response can develop in chimpanzees after an inoculation of a single HIV variant. This finding suggests that a common, less immunodominant determinant(s) is present on a single HIV strain which could induce group-specific antibodies during viral infection and replication. The identification of this group-specific epitope and the induction of analogous immunity may be relevant to vaccine development against human acquired immunodeficiency syndrome.     

Lack of transmission of human immunodeficiency virus from infected to uninfected chimpanzees

Four uninfected chimpanzees were each housed in separate cages with an HIV-infected chimpanzee for six to twenty-nine months. Despite close daily contact, all uninoculated chimpanzees remained seronegative for HIV, and virus was never isolated from peripheral blood mononuclear cells of the uninfected chimpanzees. These data indicate that the probability of transmission from infected animals to humans is extremely low and also provide supportive evidence for lack of transmission of HIV by casual contact.     

Superinfection of a chimpanzee with a second strain of human immunodeficiency virus.

Two chimpanzees, one (C-499) infected with the acquired immunodeficiency syndrome-associated retrovirus type 2 (ARV-2) strain of human immunodeficiency virus (HIV) and one (C-560) infected with the lymphadenopathy-associated virus type 1 (LAV-1) strain of HIV, were inoculated with approximately 10(4) tissue culture infective doses of the reciprocal strain. At the time of the second inoculation, both chimpanzees had high titers of HIV-specific antibodies, including antibodies that neutralized both virus strains. After inoculation of the second strain of HIV, the antibody titers in both chimpanzees increased 4- to 10-fold, and in one chimpanzee (C-499), the numbers of infectious peripheral blood mononuclear cells (PBMC) increased 1,000-fold to levels that are comparable with those observed after primary HIV infections. By restriction enzyme analysis of virus recovered from PBMC, both ARV-2 and LAV-1 were identified in C-499, thus demonstrating that superinfection had occurred.     

T-cell responses to human immunodeficiency virus (HIV) and its recombinant antigens in HIV-infected chimpanzees.

Peripheral blood lymphocytes from chimpanzees infected for 3 months to more than 3 years with human immunodeficiency virus (HIV) had normal T-cell proliferative responses after stimulation with a variety of recall antigens and mitogens, indicating that HIV infection does not cause detectable immunological impairment in chimpanzees. This finding contrasts with that obtained in HIV-infected humans, who often have impaired T-cell reactivity. Peripheral blood lymphocytes from most HIV-infected chimpanzees that were studied also had strong proliferative responses to purified HIV as well as to HIV envelope glycoproteins isolated from the virus, to recombinant HIV envelope glycoproteins gp120 and gp41, and to HIV gag protein p24. The HIV-specific T-cell responses in HIV-infected chimpanzees may contribute to prevention of the development of acquired immunodeficiency syndrome in this species.     

Proliferative and cytotoxic T cells to AIDS virus glycoproteins in chimpanzees immunized with a recombinant vaccinia virus expressing AIDS virus envelope glycoproteins

PBL from chimpanzees immunized with a recombinant vaccinia virus that expresses HIV envelope glycoproteins ("env"), were found to proliferate after stimulation with HIV or with "env". Furthermore, CTL clones lytic for autologous target cells expressing HIV envelope glycoproteins were generated after stimulation of the chimpanzees' PBL with "env", indicating that immunization of these primates with a recombinant vaccinia virus primes HIV-specific CTL and also that HIV envelope glycoproteins serve as target antigens for CTL.     

A CD4 domain important for HIV-mediated syncytium formation lies outside the virus binding site

HIV infection of chimpanzees results in a chronic viremia unaccompanied by the ultimately fatal immunodeficiency that marks HIV infection in man. We show here that expression of HIV envelope proteins allows syncytium formation between cells expressing human but not chimpanzee or macaque CD4. We find that the CD4 sequences regulating cell fusion lie outside the recognized virus binding site; in the simplest exchange, chimpanzee CD4 bearing human residue 87 supports syncytium formation, while human CD4 bearing chimpanzee residue 87 does not. Neither the equilibrium nor the forward rate constants for HIV-CD4 association are affected by substitution at position 87. Infection of human cells expressing chimpanzee CD4 is insensitive to lysosomotropic agents, suggesting that viral penetration under these circumstances does not require endocytosis. The benign course of HIV infection in chimpanzees may reflect the failure of the host to support direct cell to cell transmission of the virus.     

The biology of human immunodeficiency virus-1 IIIB infection in the chimpanzee: in vivo and in vitro correlations

Human immunodeficiency virus (HIV)-1 IIIB infection of chimpanzees leads to a compartmentalized, nonpathogenic in vivo and in vitro relationship with the virus. The absence of an acquired immunodeficiency syndrome (AIDS)-like disease in over 100 chimpanzees persistently infected may be related to some or all of the findings reported here. Further characterizing these possible host adapative mechanisms may be critical in both understanding pathogenesis, as well as elucidating novel mechanisms for therapeutic and/or the preventive strategies for AIDS in humans.     

Proliferative T-cell response to HIV envelope glycoprotein in immunized and infected primates and human beings

Human immunodeficiency virus (HIV)-specific helper T-cell response was studied in human subjects and nonhuman primates either infected with HIV or immunized with different HIV protein preparations. A strong group-specific T-cell response involving T-cell proliferation and lymphokine secretion was observed in immunized chimpanzees and rhesus monkeys as well as HIV-infected chimpanzees and gibbons. HIV-infected people demonstrated a low or no HIV-specific T-cell response. In contrast, five of 14 HIV antibody-negative sexual partners of HIV-infected men recognized one or more T-cell epitopes in the envelope glycoprotein of HIV.     

Genetic subspecies diversity of the chimpanzee CD4 virus-receptor gene

Chimpanzees are naturally and asymptomatically infected by simian immunodeficiency virus (SIV). Pathogenic properties of SIV/HIV vary and differences in susceptibility and pathogenicity of SIV/HIV depend in part on host-specific factors such as virus-receptor/co-receptor interactions. Since CD4 plays a primary role in virus binding and since SIVcpz have been found only in two African chimpanzee subspecies, we characterized the genetic diversity of CD4 receptors in all four recognized subspecies of chimpanzees. We found noticeable variation in the first variable region V1 of CD4 and in intron six among the subspecies of chimpanzees. We found the CD4 receptor to be conserved in individuals belonging to the P. t. verus subspecies and divergent from the other three subspecies, which harbored highly variable CD4 receptors. The CD4 receptor of chimpanzees differed from that of humans. We question whether the observed diversity can explain the species-specific differences in susceptibility to and pathogenicity of SIV/HIV.     

Telomere dynamics in HIV-1 infected and uninfected chimpanzees measured by an improved method based on high-resolution two-dimensional calibration of DNA sizes

We developed an improved method for accurately measuring telomere lengths based on two-dimensional calibration of DNA sizes combined with pulsed field electrophoresis and quantitative analysis of high-resolution gel images. This method was used to quantify the length of telomeres in longitudinal samples of peripheral blood mononuclear cells (PBMCs) from five chimpanzees infected with human immunodeficiency virus type 1 (HIV-1) and three uninfected animals, 14 to 27 years of age. The average length of the telomere restriction fragments (TRF) of infected and uninfected chimpanzees were 11.7 ± 0.25 kbp, and 11.6 ± 0.61 kbp, respectively, and were about 1 kbp and 3 kbp longer than those of human infants and 30 year old adults, respectively. There was a trend of a slight decrease (30–60 bp per year) in the TRF of two HIV infected chimpanzees over 30–35 months, while the TRF of one naive chimpanzee slightly increased over 20 months. Although the number of chimpanzees in this study is small and no statistically significant linear dependencies on time were observed, it appears that in chimpanzees, rates of shortening of the TRF are comparable or smaller than in adult humans and are not significantly affected by HIV-1 infection, which may be related to the inability of HIV-1 to cause disease in these animals.     

Persistent infection of chimpanzees with human T-lymphotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome.

The lymphadenopathy-associated virus (LAV) prototype strain of human T-lymphotropic virus type III/LAV was transmitted to juvenile chimpanzees with no prior immunostimulation by (i) intravenous injection of autologous cells infected in vitro, (ii) intravenous injection of cell-free virus, and (iii) transfusion from a previously infected chimpanzee. All five animals that received more than one 50% tissue culture infective dose were persistently infected with LAV or chimpanzee-passaged LAV for up to 18 months. During this time they developed no illnesses, but they exhibited various degrees of inguinal and axillary lymphadenopathy and significant reductions in rates of weight gain. Detailed blood chemistry and hematologic evaluations revealed no consistent abnormalities, with the exception of immunoglobulin G (IgG) hypergammaglobulinemia, which became apparent in one animal 6 months postinfection and continued at more than 1 year postinfection. Transient depressions followed by increases in the numbers of T4 cells to levels greater than normal were observed in all animals after virus inoculation. However, the number of LAV-infected peripheral blood cells decreased with time after infection. Results of enzyme immunoassays showed that all infected animals seroconverted to IgG anti-LAV within 1 month postinfection and that antibody titers remained high throughout the period of observation. In contrast, only three of the five LAV-infected chimpanzees had detectable IgM antibody responses, and these preceded IgG-specific serum antibodies by 1 to 2 weeks. Virus morphologically and serologically identical to LAV was isolated from peripheral blood mononuclear cells of all infected animals at all times tested and from bone marrow cells taken from one animal 8 months after infection. One chimpanzee that was exposed to LAV only by sharing a cage with an infected chimpanzee developed lymphadenopathy and an IgM response to LAV, both of which were transient; however, no persistent IgG antibody response to LAV developed, and no virus was recovered from peripheral blood cells during a year of follow-up. Thus, LAV readily infected chimpanzees following intravenous inoculation and persisted for extended periods despite the presence of high titers of antiviral antibodies. However, the virus was not easily transmitted from infected to uninfected chimpanzees during daily cage contact.     

Nonclassical mucosal antibodies predominate in genital secretions of HIV-1 infected chimpanzees

To identify mucosal immunity in HIV-infected chimpanzees, IgG, IgA, and IgM from plasma, saliva, rectal swabs, vaginal washes, semen, and urethral washes were tested from four male and three female HIV-1_IIIB infected chimpanzees. The level of HIV infections in the seven chimpanzees were classified as high, intermediate and low depending on the number of HIV-1 infected cells per 10⁷ peripheral blood mononuclear cells (PBMC). One male chimpanzee had a relatively high viral load, two males and two females had moderate viral loads and one male and one female had low levels of infection. All seven animals had plasma antibody. The principal finding was that nonclassical mucosal antibodies of the IgG isotype were the predominant antibody in the saliva, rectal swabs, vaginal washes, semen, and urethral washes of infected animals. All plasma and mucosal samples were negative for IgM antibodies. The results show that HIV-1 specific IgG responses and not sIgA predominate at mucosal surfaces of HIV-1_IIIB infected chimpanzees. A trend was observed in which high viral loads correlated with high plasma IgG, IgA and sIgA titers. An overall correlation between relatively high virus loads and high amounts of mucosal IgG was also found.     

Natural and iatrogenic factors in human immunodeficiency virus transmission

In the light of the evidence and discussion presented during The Royal Society Discussion Meeting it seems to me that the oral polio vaccine (OPV) hypothesis for the origins of human immunodeficiency virus (HIV) and the acquired immune deficiency syndrome epidemic is less tenable now than one year earlier. The OPV hypothesis does not accord with HIV phylogenetic studies: the geographical correlation has been challenged; the testimony of those directly involved with OPV trial vaccines denies the use of chimpanzees, corroborating tests on the still-available vials of the CHAT vaccines, which contain neither simian immunodeficiency virus nor chimpanzee DNA. Yet one lesson to be learned from considering OPV as a source of HIV is how plausibly it might have happened and how cautious we need to be over introducing medical treatments derived from animal tissues, such as live, attenuated vaccines or xenotransplantation. To cast doubt on the OPV hypothesis is not to dismiss entirely the role of iatrogenic factors in HIV transmission from chimpanzees in the first instance, in HIV adaptation to onward transmission during its early phase in humans, or in the later spread of HIV to patients, for example, with haemophilia. To reduce the argument over the origins of HIV to the 'OPV hypothesis' versus the 'cut-hunter hypothesis' is an oversimplistic and false antithesis. Both natural and iatrogenic transmission of many retroviruses, including HIV, have been thoroughly documented and are not mutually exclusive. Exactly how, when and where the first human(s) became infected with the progenitor of HIV-1 group M, which gave rise to the pandemic strain, is likely, however, to remain a matter of conjecture.     

A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees

Three percent of the world's population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.     

Appropriate conditions for maintenance of chimpanzees in studies with blood-borne viruses: an epidemiologic and psychosocial perspective

Based on the know epidemiology of the viruses that account for the bulk of the need for chimpanzees in biomedical research--hepatitis B virus (HBV), non-A, non-B (NANB) hepatitis virus, and human immunodeficiency virus (HIV)--as well as the psychosocial needs of this species, requirements for appropriate isolation conditions for these animals have been reviewed. We believe that animals should generally be housed in groups of at least two in the same cage, and that cages encased in solid-walled isolator boxes for housing of single chimpanzees are unnecessary for virologically adequate isolation for studies of HBV, NANB and HIV, and cause sensory and psychosocial deprivation, which contravenes their psychological well-being.     

In vitro susceptibility of T lymphocytes from chimpanzees (Pan troglodytes) to human herpesvirus 6 (HHV-6): a potential animal model to study the interaction between HHV-6 and human immunodeficiency virus type 1 in vivo.

The in vitro susceptibility of several nonhuman primate species to human herpesvirus 6 (HHV-6) was investigated. Only peripheral blood mononuclear cells from chimpanzees (Pan troglodytes) were found permissive to productive infection by HHV-6, indicating that the host range of HHV-6, albeit limited, may not be restricted to Homo sapiens. However, natural HHV-6 infection in chimpanzees, as well as in the other species tested, could not be documented by serological analysis. As previously observed with human cells, HHV-6 infection of chimpanzee peripheral blood mononuclear cells was highly cytopathic and the infected cells exhibited phenotypic features of activated T lymphocytes. Although in humans the majority of HHV-6-infected lymphocytes displayed the CD4 antigen, in chimpanzees a mixed CD4+ and CD8+ phenotype was observed. HHV-6 was also shown to productively coinfect individual chimpanzee T cells with human immunodeficiency virus type 1, resulting in an accelerated induction of cytopathicity. In light of these findings, we propose the utilization of chimpanzees as a potential animal model system to investigate the in vivo interaction between HHV-6 and human immunodeficiency virus type 1 and its relevance to the development of acquired immune deficiency syndrome.     

Vaccine-induced protection of chimpanzees against infection by a heterologous human immunodeficiency virus type 1.

The extraordinary genetic diversity of human immunodeficiency virus type 1 (HIV-1) is a major problem to overcome in the development of an effective vaccine. In the most reliable animal model of HIV-1 infection, chimpanzees were immunized with various combinations of HIV-1 antigens, which were derived primarily from the surface glycoprotein, gp160, of HIV-1 strains LAI and MN. The immunogens also included a live recombinant canarypox virus expressing a gp160-MN protein. In one experiment, two chimpanzees were immunized multiple times; one animal received antigens derived only from HIV-1LAI, and the second animal received antigens from both HIV-1LAI and HIV-1MN. In another experiment, four chimpanzees were immunized in parallel a total of five times over 18 months; two animals received purified gp160 and V3-MN peptides, whereas the other two animals received the recombinant canarypox virus and gp160. At 3 months after the final booster, all immunized and naive control chimpanzees were challenged by intravenous inoculation of HIV-1SF2; therefore, the study represented an intrasubtype B heterologous virus challenge. Virologic and serologic follow-up showed that the controls and the two chimpanzees immunized with the live recombinant canarypox virus became infected, whereas the other animals that were immunized with gp160 and V3-MN peptides were protected from infection. Evaluation of both cellular and humoral HIV-specific immune responses at the time of infectious HIV-1 challenge identified the following as possible correlates of protection: antibody titers to the V3 loop of MN and neutralizing antibody titers to HIV-1MN or HIV-1LAI, but not to HIV-1SF2. The results of this study indicate that vaccine-mediated protection against intravenous infection with heterologous HIV-1 strains of the same subtype is possible with some immunogens.     

In vivo protective anti-HIV immune responses in non-human primates through DNA immunization

Abstract: An effective immune response involves the specific recognition of and elimination of an infectious organism at multiple levels. In this context DNA immunization can present functional antigenic proteins to the host for recognition by all arms of the immune system, yet provides the opportunity to delete any genes of the infectious organism which code for antigens or pieces of antigens that may have deleterious effects. Our group has developed the use of nucleic acid immunization as a possible method of vaccination against Human immunodeficiency virus type 1 (HIV-1) [1,2,3,10,11,12]. Sera from non-human primates immunized with DNA vectors that express the envelope proteins from HIV-1 contain antibodies specific to the HIV-1 envelope. These sera also neutralize HIV-1 infection in vitro and inhibit cell to cell infection in tissue culture. Analysis of cellular responses is equally encouraging. T cell proliferation as well as cytotoxic T cell lysis of relevant env expressing target cells were observed. In addition, evidence that DNA vaccines are capable of inducing a protective response against live virus was demonstrated using a chimeric SIV/HIV (SHIV) challenge in vaccinated cynomologous macaques. We found that nucleic acid vaccination induced protection from challenge in one out of four immunized cynomolgus macaques and viral load was lower in the vaccinated group of animals versus the control group of animals. These data encouraged us to analyze this vaccination technique in chimpanzees, the most closely related animal species to man. We observed the induction of both cellular and humoral immune responses with a DNA vaccine in chimpanzees. These studies demonstrate the utility of this technology to induce relevant immune responses in primates which may ultimately lead to effective vaccines.     

Replication of human immunodeficiency virus in two T-cell lines and their application as antigens for immunofluorescence

Two T-cell lines, TALL-1 and CCRF-CEM, were infected with human immunodeficiency virus (HIV), strain LAV, to explore the time course of the appearance of various virus specific antigens, and to establish an antibody assay system by indirect immunofluorescence (IF). These cells were infected with LAV at two different input multiplicity of infection (MOI). Antigens were tested by Western blot analysis (WB) and IF. Antigens for WB were extracted from the infected cells at various times after infection, but pooled sera of American HIV carriers could not recognize gp41 or gp160. Antigen expression was highest in CCRF-CEM, but, as the antigen for IF, TALL-1 infected at the MOI of 8.0 was the most suitable 7 days after infection, because it includes a fairly large number of uninfected cells, which served as the internal control.