Harold M. McClure

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.

Nonpathogenic SIV Infection of Sooty Mangabeys Is Characterized by Limited Bystander Immunopathology Despite Chronic High-Level Viremia

HIV-infected humans and SIV-infected rhesus macaques who remain healthy despite long-term infection exhibit exceptionally low levels of virus replication and active antiviral cellular immune responses. In contrast, sooty mangabey monkeys that represent natural hosts for SIV infection do not develop AIDS despite high levels of virus replication and limited antiviral CD8(+) T cell responses. We report here that SIV-infected mangabeys maintain preserved T lymphocyte populations and regenerative capacity and manifest far lower levels of aberrant immune activation and apoptosis than are seen in pathogenic SIV and HIV infections. These data suggest that direct consequences of virus replication alone cannot account for progressive CD4(+) T cell depletion leading to AIDS. Rather, attenuated immune activation enables SIV-infected mangabeys to avoid the bystander damage seen in pathogenic infections and protects them from developing AIDS.

Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations.

Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.

A substantial risk in using live attenuated, multiply deleted viruses as vaccines against AIDS is their potential to induce AIDS. A mutant of the simian immunodeficiency virus (SIV) with large deletions in nef and vpr and in the negative regulatory element induced AIDS in six of eight infant macaques vaccinated orally or intravenously. Early signs of immune dysfunction were seen in the remaining two offspring. Prolonged follow–up of sixteen vaccinated adult macaques also showed resurgence of chronic viremia in four animals: two of these developed early signs of disease and one died of AIDS. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines built on similar prototypes may cause AIDS.

Divergent Host Responses during Primary Simian Immunodeficiency Virus SIVsm Infection of Natural Sooty Mangabey and Nonnatural Rhesus Macaque Hosts

ABSTRACT To understand how natural sooty mangabey hosts avoid AIDS despite high levels of simian immunodeficiency virus (SIV) SIVsm replication, we inoculated mangabeys and nonnatural rhesus macaque hosts with an identical inoculum of uncloned SIVsm. The unpassaged virus established infection with high-level viral replication in both macaques and mangabeys. A species-specific, divergent immune response to SIV was evident from the first days of infection and maintained in the chronic phase, with macaques showing immediate and persistent T-cell proliferation, whereas mangabeys displayed little T-cell proliferation, suggesting subdued cellular immune responses to SIV. Importantly, only macaques developed CD4+-T-cell depletion and AIDS, thus indicating that in mangabeys limited immune activation is a key mechanism to avoid immunodeficiency despite high levels of SIVsm replication. These studies demonstrate that it is the host response to infection, rather than properties inherent to the virus itself, that causes immunodeficiency in SIV-infected nonhuman primates.

Critical Role for Env as well as Gag-Pol in Control of a Simian-Human Immunodeficiency Virus 89.6P Challenge by a DNA Prime/Recombinant Modified Vaccinia Virus Ankara Vaccine

ABSTRACT Cellular immune responses against epitopes in conserved Gag and Pol sequences of human immunodeficiency virus type 1 have become popular targets for candidate AIDS vaccines. Recently, we used a simian-human immunodeficiency virus model (SHIV 89.6P) with macaques to demonstrate the control of a pathogenic mucosal challenge by priming with Gag-Pol-Env-expressing DNA and boosting with Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (rMVA). Here we tested Gag-Pol DNA priming and Gag-Pol rMVA boosting to evaluate the contribution of anti-Env immune responses to viral control. The Gag-Pol vaccine raised frequencies of Gag-specific T cells similar to those raised by the Gag-Pol-Env vaccine. Following challenge, these rapidly expanded to counter the challenge infection. Despite this, the control of the SHIV 89.6P challenge was delayed and inconsistent in the Gag-Pol-vaccinated group and all of the animals underwent severe and, in most cases, sustained loss of CD4+ cells. Interestingly, most of the CD4+ cells that were lost in the Gag-Pol-vaccinated group were uninfected cells. We suggest that the rapid appearance of binding antibody for Env in Gag-Pol-Env-vaccinated animals helped protect uninfected CD4+ cells from Env-induced apoptosis. Our results highlight the importance of immune responses to Env, as well as to Gag-Pol, in the control of immunodeficiency virus challenges and the protection of CD4+ cells.

Sensitive and robust one-tube real-time reverse transcriptase-polymerase chain reaction to quantify SIV RNA load: comparison of one- versus two-enzyme systems.

Plasma viral RNA load is a key parameter in disease progression of lentiviral infections. To measure simian immunodeficiency virus (SIV) RNA loads, we have established a quantitative one-tube assay based on TaqMan chemistry. This real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has advantages compared with previous methods, such as higher sensitivity, shorter time consumption, and low risk of cross-contamination. The sensitivity of the assay was optimized by comparing different enzyme systems. The one-enzyme protocol using rTth DNA polymerase was superior to two assays employing two enzymes. It detects 100% of the samples containing four copies of RNA transcript and allows quantification of viral RNA loads over an 8-log unit dynamic range. As few as 50 copies per milliliter of plasma can be detected within RNA extracted from 140 microl of plasma. This is especially relevant in studies employing neonatal macaques, from which only small volumes of blood can be sampled, and in studies in which low viral RNA loads are expected. Because of the use of rTth DNA polymerase, DNA contamination can be avoided by carryover prevention with uracil N-glycosylase (UNG). We demonstrate that for optimization of real-time PCR sensitivity, not only concentrations of different reagents but also different enzyme systems must be evaluated. Our assay facilitates and enhances the quantification of plasma RNA loads, a critical parameter for many studies, including evaluations of vaccine candidates or antiviral regimens.

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.

Complete Protection of Neonatal Rhesus Macaques against Oral Exposure to Pathogenic Simian-Human Immunodeficiency Virus by Human Anti-HIV Monoclonal Antibodies

Because milk-borne transmission of human immunodeficiency virus (HIV) diminishes the benefits of perinatal antiviral drug therapy in developing countries, we have developed a new strategy to prevent postnatal and, possibly, intrapartum virus transmission in a primate model. Eight neonatal rhesus macaques were exposed orally to pathogenic simian-human immunodeficiency virus (SHIV); 4 neonates were then given intramuscular postexposure prophylaxis with 3 anti-HIV human neutralizing monoclonal antibodies (nMAbs) with potent cross-clade and cross-group neutralization activity. Untreated infants experienced high viral RNA levels and CD4(+) T-cell losses and died (median survival time, 5.5 weeks). In contrast, all 4 nMAb-treated neonates were protected from infection (P=.028); their plasma, peripheral blood mononuclear cells, and lymph nodes remained virus negative for >1 year. These data are important for designing clinical trials in human neonates and have general implications for AIDS vaccine development, as the epitopes recognized by the 3 nMAbs are conserved among diverse primary isolates.

Different Patterns of Immune Responses but Similar Control of a Simian-Human Immunodeficiency Virus 89.6P Mucosal Challenge by Modified Vaccinia Virus Ankara (MVA) and DNA/MVA Vaccines

ABSTRACT Recently we demonstrated the control of a mucosal challenge with a pathogenic chimera of simian and human immunodeficiency virus (SHIV-89.6P) by priming with a Gag-Pol-Env-expressing DNA and boosting with a Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (DNA/MVA) vaccine. Here we evaluate the ability of the MVA component of this vaccine to serve as both a prime and a boost for an AIDS vaccine. The same immunization schedule, MVA dose, and challenge conditions were used as in the prior DNA/MVA vaccine trial. Compared to the DNA/MVA vaccine, the MVA-only vaccine raised less than 1/10 the number of vaccine-specific T cells but 10-fold-higher titers of binding antibody for Env. Postchallenge, the animals vaccinated with MVA alone increased their CD8 cell numbers to levels that were similar to those seen in DNA/MVA-vaccinated animals. However, they underwent a slower emergence and contraction of antiviral CD8 T cells and were slower to generate neutralizing antibodies than the DNA/MVA-vaccinated animals. Despite this, by 5 weeks postchallenge, the MVA-only-vaccinated animals had achieved as good control of the viral infection as the DNA/MVA group, a situation that has held up to the present time in the trial (48 weeks postchallenge). Thus, MVA vaccines, as well as DNA/MVA vaccines, merit further evaluation for their ability to control the current AIDS pandemic.