Formaldehyde-treated, heat-inactivated virions with increased human immunodeficiency virus type 1 env can be used to induce high-titer neutralizing antibody responses.

The lack of success of subunit human immunodeficiency virus (HIV) type 1 vaccines to date suggests that multiple components or a complex virion structure may be required. We hypothesized that the failure of current vaccine strategies to induce protective antibodies is linked to the inability of native envelope structures to readily elicit these types of antibodies. We have previously reported on the ability of a formaldehyde-treated, heat-inactivated vaccine to induce modest antibody responses in animal vaccine models. We investigated here whether immunization for HIV with an envelope-modified, formaldehyde-stabilized, heat-inactivated virion vaccine could produce higher-titer and/or broader neutralizing antibody responses. Thus, a clade B vaccine which contains a single point mutation in gp41 (Y706C) that results in increased incorporation of oligomeric Env into virions was constructed. This vaccine was capable of inducing high-titer antibodies that could neutralize heterologous viruses, including those of clades A and C. These results further support the development of HIV vaccines with modifications in native Env structures for the induction of neutralizing antibody responses.

Induction of humoral immune responses following vaccination with envelope-containing, formaldehyde-treated, thermally inactivated human immunodeficiency virus type 1.

The lack of success of subunit human immunodeficiency virus type 1 (HIV-1) vaccines to date suggests that multiple components or a complex virion structure may be required. We previously demonstrated retention of the major conformational epitopes of HIV-1 envelope following thermal treatment of virions. Moreover, antibody binding to some of these epitopes was significantly enhanced following thermal treatment. These included the neutralizing epitopes identified by monoclonal antibodies 1b12, 2G12, and 17b, some of which have been postulated to be partially occluded or cryptic in native virions. Based upon this finding, we hypothesized that a killed HIV vaccine could be derived to elicit protective humoral immune responses. Shedding of HIV-1 envelope has been described for some strains of HIV-1 and has been cited as one of the major impediments to developing an inactivated HIV-1 vaccine. In the present study, we demonstrate that treatment of virions with low-dose formaldehyde prior to thermal inactivation retains the association of viral envelope with virions. Moreover, mice and nonhuman primates vaccinated with formaldehyde-treated, thermally inactivated virions produce antibodies capable of neutralizing heterologous strains of HIV in peripheral blood mononuclear cell-, MAGI cell-, and U87-based infectivity assays. These data indicate that it is possible to create an immunogen by using formaldehyde-treated, thermally inactivated HIV-1 virions to induce neutralizing antibodies. These findings have broad implications for vaccine development.

Enhanced binding of antibodies to neutralization epitopes following thermal and chemical inactivation of human immunodeficiency virus type 1.

Inactivation of viral particles is the basis for several vaccines currently in use. Initial attempts to use simian immunodeficiency virus to model a killed human immunodeficiency virus type 1 (HIV-1) vaccine were unsuccessful, and limited subsequent effort has been directed toward a systematic study of the requirements for a protective killed HIV-1 vaccine. Recent insights into HIV-1 virion and glycoprotein structure and neutralization epitopes led us to revisit whether inactivated HIV-1 particles could serve as the basis for an HIV-1 vaccine. Our results indicate that relatively simple processes involving thermal and chemical inactivation can inactivate HIV-1 by at least 7 logs. For some HIV-1 strains, significant amounts of envelope glycoproteins are retained in high-molecular-weight fractions. Importantly, we demonstrate retention of each of three conformation-dependent neutralization epitopes. Moreover, reactivity of monoclonal antibodies directed toward these epitopes is increased following treatment, suggesting greater exposure of the epitopes. In contrast, treatment of free envelope under the same conditions leads only to decreased antibody recognition. These inactivated virions can also be presented by human dendritic cells to direct a cell-mediated immune response in vitro. These data indicate that a systematic study of HIV-1 inactivation, gp120 retention, and epitope reactivity with conformation-specific neutralizing antibodies can provide important insights for the development of an effective killed HIV-1 vaccine.

Potential contributions of viral envelope and host genetic factors in a human immunodeficiency virus type 1-infected long-term survivor.

The lack of clinical progression in some individuals despite prolonged human immunodeficiency virus type 1 (HIV-1) infection may result from infection with less-pathogenic viral strains. To address this question, we examined the HIV-1 envelope protein from a donor with a low viral burden, stable CD4(+) T-lymphocyte counts, and little evidence of CD8(+) T-cell expansion, activation, or immune activity. To avoid potential changes in envelope function resulting from selection in vitro, envelope clones were constructed by using viral RNA isolated from uncultured peripheral blood mononuclear cells (PBMC). The data showed that recombinant viruses containing envelope sequences derived from RNA isolated from patient PBMC replicated poorly in primary CD4(+) T cells but demonstrated efficient growth in macrophages. The unusual phenotype of these viruses could not be explained solely by differential utilization of coreceptors since the chimeric viruses, as well as an uncloned isolate obtained from the same visit date, can utilize CCR5. In addition, the donor's own cells appeared resistant to infection with chimeric viruses containing autologous envelope sequences. Genotype analysis revealed that the donor was heterozygous for the previously described 32-bp deletion in CCR5 which may be linked with prolonged survival in HIV-1-infected individuals. These data suggest that the changes in envelope sequences confer properties of viral attenuation, which together with the CCR5 +/Delta32 genotype could account for the long-term survival of this patient.

Phenotypically defined memory CD4+ cells are not selectively decreased in chronic HIV disease.

Simultaneous measurements of phenotypically defined memory CD4+ cells and in vitro proliferation to three recall antigens (Ags; tetanus toxoid, influenza, and Candida albicans) were performed in 53 HIV-seropositive subjects and 39 HIV-seronegative controls. The results indicate that the low proliferative responses to recall Ags of those who were HIV infected could be partly, but not fully, explained by a decrease of phenotypically defined memory CD4+ cells. This is, to our knowledge, the first report of experiments that simultaneously measured memory CD4+ cell numbers and function and then examined whether the low responses observed in seropositive subjects could be explained by low numbers of phenotypically defined memory CD4+ cells. A central finding of the study, which argues against prevailing dogma, was that within the CD4+ lymphocyte population, the proportion of cells displaying the memory phenotype was not selectively decreased in HIV-seropositive subjects as compared with the proportion of these cells in seronegative homosexual controls. An entirely new finding of the study was that AIDS patients, many of whom were unresponsive to all three recall Ags tested, actually had a significant increase in the proportion of CD4+ cells with the memory phenotype, and this fraction approached 100% in subjects with CD4+ cell numbers that were near zero. A final observation of the study, possible because some patients were on zidovudine (ZDV), was that there was no evidence that ZDV treatment led to an increased proliferative response to recall Ags in vivo. An in vitro study also found no effect of ZDV, dideoxycytidine (ddC), or azido-dideoxyuridine (AZU) on proliferative responses to recall Ags.

Cell-mediated immune response to human immunodeficiency virus (HIV) type 1 in seronegative homosexual men with recent sexual exposure to HIV-1.

Although human immunodeficiency virus (HIV) type 1 infection is efficiently transmitted by sexual intercourse, some individuals whose sexual behavior places them at extremely high risk for infection have nevertheless remained HIV-1-seronegative. An investigation was undertaken to determine whether such individuals have circulating T helper cells that are sensitized to HIV-1. Five very high risk men who had recent sexual exposure to HIV-1 were studied. Peripheral blood mononuclear cells from all 5 produced interleukin (IL)-2 in culture in response to synthetic amphipathic HIV-1 envelope peptides. One of the 5 high-risk men has subsequently seroconverted, while 4 have remained seronegative. All were initially culture-negative, and those who have remained seronegative were also virus-negative by polymerase chain reaction (PCR) testing 10 months after they were first studied. These results demonstrate that a cell-mediated immune response to HIV-1 can be detected in the absence of a humoral immune response in individuals recently exposed to HIV-1. Furthermore, IL-2 production by T cells in response to synthetic peptides may be a more sensitive test for exposure to HIV-1 than antibody, lymphoproliferation, or PCR tests.