Comprehensive analyses of a unique HIV-1-infected nonprogressor reveal a complex association of immunobiological mechanisms in the context of replication-incompetent infection

We recently demonstrated that a unique HIV-1-infected nonprogressor was infected with a nonevolving replication-incompetent HIV-1 strain, showing a total absence of viral evolution in vivo. Potent immune responses against HIV-1 were observed in his PBMC, despite an apparent lack of viral replication for at least 8 years. His PBMC resisted superinfection with CCR5, CXCR4, and dual-tropic HIV-1 strains, although highly purified CD4+ T cells supported infection, but without any visible cytopathic effect. Potent noncytolytic CD8+ T cell antiviral activity was shown to protect his PBMC from productive infection. This activity was not mediated by several known chemokines or IFN-gamma, which were produced at high levels after PHA activation of his CD8+ T cells, indicating the action of other CAF-like CD8 factors. This antiviral activity was a memory response, induced by HIV-specific stimulation to similar levels observed by PHA stimulation, but absent in ex vivo resting T cells. Immunological mechanisms associated with this antiviral suppressive activity included vigorous Gag-specific helper T cell proliferative responses and high-level IFN-gamma release by both CD4 and CD8 T cells. These responses were broadly directed against multiple Gag epitopes, both previously reported and some novel epitopes. Strong HIV-specific helper T cell function was also associated with strong neutralizing antibodies. Understanding how to induce these protective immune responses in other individuals could provide a major step forward in the design of effective immunotherapies or vaccines against HIV infection.     

Adenoviral gene delivery for HIV-1 vaccination

The AIDS epidemic continues to spread throughout nations of Africa and Asia and is by now threatening to undermine the already frail infrastructure of developing countries in Sub-Saharan Africa that are hit the hardest. The only option to stem this epidemic is through inexpensive and efficacious vaccines that prevent or at least blunt HIV-1 infections. Despite decades of pre-clinical and clinical research such vaccines remain elusive. Most anti-viral vaccines act by inducing protective levels of virus-neutralizing antibodies. The envelope protein of HIV-1, the sole target of neutralizing antibodies, is constantly changing due to mutations, B cell epitopes are masked by heavy glycosylation and the protein's structural unfolding upon binding to its CD4 receptor and chemokine co-receptors. Efforts to induce broadly cross-reactive virus-neutralizing antibodies able to induce sterilizing or near sterilizing immunity to HIV-1 have thus failed. Studies have indicated that cell-mediated immune responses and in particular CD8+ T cell responses to internal viral proteins may control HIV-1 infections without necessarily preventing them. Adenoviral vectors expressing antigens of HIV-1 are eminently suited to stimulate potent CD8+ T cell responses against transgene products, such as antigens of HIV-1. They performed well in pre-clinical studies in rodents and nonhuman primates and are currently in human clinical trials. This review summarizes the published literature on adenoviral vectors as vaccine carriers for HIV-1 and discusses advantages and disadvantages of this vaccine modality.     

Changes in natural immunity during the course of HIV-1 infection.

The role of natural killer (NK) and lymphokine-activated killer (LAK) cell-mediated cytotoxicity in AIDS has yet to be established. The objective of this study was to determine inducible LAK cell responses at different stages of HIV-1 infection, and specifically to establish the participation of CD8 lymphocytes in these responses. Peripheral blood lymphocytes (PBL) were isolated from healthy seronegative (CDC-0) subjects and HIV-1+ individuals who were clinically asymptomatic (Centre for Disease Control group 2, CDC-2) or symptomatic (CDC-4) with regard to secondary opportunistic infection (OI). LAK cells were generated upon incubation of PBL with IL-2 and their cytolysis of K562 and U-937 targets was determined using chromium release assays. The role of CD8+ lymphocytes as progenitors and effectors of these LAK cell responses was determined by immunomagnetic depletion of CD8+ cells from precursor PBL and LAK cells, respectively. LAK cell-mediated cytotoxicities in HIV-1-infected individuals were reduced compared with seronegative controls without any corresponding changes in the relative proportions of CD56+ (NK) cells among groups. Depletions of CD8+ subsets from either PBL or LAK cells dramatically reduced total LAK cytotoxic responses and LAK activities per unit CD56+ cell in the OI-/CDC-2 seropositive population. No corresponding changes in LAK activities in seronegative control or HIV+/OI+/CDC-4 groups were observed. Levels of LAK activity against K562 targets in CDC-0/HIV- and CDC-4/HIV+ groups correlated with the percentage of CD56+ LAK cells; corresponding LAK activity in the CDC-2/HIV+ group correlated with the percentage of both CD56+ and CD8+ subsets. These findings suggest that adaptive changes in non-MHC restricted cytotoxic responses occur in HIV-1 individuals at early stages post-HIV infection, before the onset of opportunistic infection.     

CD11a/CD18 (LFA-1) epitopes involved in syncytium formation among CD4+ T-cells following cell free HIV-1 infection.

Like particular monoclonal antibodies to CD4, monoclonal antibodies to epitopes localized at the top of the alpha-subunit (CD11a) and at the stem of the beta-subunit (CD18) of the lymphocyte function associated antigen-1 (LFA-1) block syncytium formation when added to CD4+ T-cells before or shortly after (5 min) cell free HIV-1 is added. This indicates involvement of LFA-1 epitopes in the early stage of cell-free infection. Syncytium formation is still blocked by CD4 antibodies when added two hours after virus adsorption, CD11a and CD18 antibodies are ineffective at this late stage. Radio-immune precipitation experiments in acutely infected T-cells with the appropriate CD11a and CD18 antibodies suggested a link between CD18 and CD4 in the cell lines studied, possibly mediated by the CD3/TCR complex. In chronically infected cells, where little CD4 is expressed, LFA-1 antibodies still precipitate the viral envelope, pointing to a direct LFA-1 envelope interaction in that late stage of infection. Our results indicate that a limited number of LFA-1 epitopes is involved in syncytium formation among T-cells, none of which is required for virus entry in the cell or virus spread in the culture.     

Study of CD4+CD8+ double positive T-lymphocyte phenotype and function in Indian patients infected with HIV-1

CD4+CD8+ double positive T cells represent a minor peripheral blood lymphocyte population. CD4+ expression on CD8+ T cells is induced following cellular activation, and as chronic HIV-1 infection is associated with generalized immune activation, double positive T cells studies have become necessary to understand the immunopathology of human immunodeficiency virus (HIV). The frequency of double positive T cells in persons infected with HIV was studied in comparison to uninfected controls. Further, the expression of CD38, HLA-DR, and programmed death (PD)-1 on these cells were ascertained. HIV-1 specific double positive T cells were also studied for their cytokine secretory ability and phenotype. A significantly higher double positive cell population was observed in the patients with advanced HIV disease (CD4+ T cell counts below 200 cells/μl), as compared to patients with CD4+ T cell counts above 500 cells/μl. Double positive T cells from patients with symptomatic HIV disease had a significantly increased activation and exhaustion levels, compared to asymptomatic subjects and to single positive T cells from the same subjects. HIV-1 specific double positive T cells showed further increase in CD38 and PD-1 expression levels. The proportion of CD38 and PD-1 expressing total and HIV-1 specific double positive T cells correlated positively with HIV-1 plasma viremia and negatively with CD4+ T cell counts. HIV infection results in a marked increase of double positive T cell population, and this cell population shows higher level of activation and exhaustion (increased PD-1 expression) compared to the single positive CD4+ and CD8+ T cells.     

CD4+ T cell targeting of human immunodeficiency virus type 1 (HIV-1) peptide sequences present in vivo during chronic, progressive HIV-1 disease

We previously detected HIV-1 Gag-specific CD4+ T cells recognizing reference strain viral epitopes in subjects with progressive, chronic infection. To test whether these CD4+ T cells persist in vivo by failing to recognize autologous HIV-1 epitopes, we compared autologous plasma HIV-1 p24 nucleotide sequences with targeted HXB.2 strain Gag p24 CD4+ T cell epitopes in nine chronically infected, untreated subjects. In five responding subjects, 10 of 26 HXB.2 strain p24 peptides targeted by CD4+ T cells exactly matched autologous plasma viral sequences. Four subjects with plasma viral loads >100,000 copies/mL had no measurable p24-specific CD4+ T cell responses despite carrying HIV-1 strains that matched HXB.2 sequences at predicted epitopes. These results show that HIV-1-specific CD4+ T cells can persist in chronic HIV-1 infection despite recognition of epitopes present in vivo. However, with high-level in vivo HIV-1 replication, CD4+ T cells targeting autologous HIV-1 may be non-responsive or absent.     

CD4-independent inhibition of lymphocyte proliferation mediated by HIV-1 envelope glycoproteins.

The cytopathic effects of HIV-1 produced by direct infection of human T cells do not account for the disproportionate loss of CD4-positive lymphocytes during the course of HIV infection. Previous studies have demonstrated the inhibition of uninfected human T cell activation and proliferation by the HIV-1 envelope glycoproteins, presumably due to gp120-CD4 interactions. To examine the ability of HIV-1 to inhibit T cell proliferation in the absence of both direct infection and gp120-CD4 interactions, we tested the effect of HIV-1 on mouse T cell proliferation. Culture media containing HIV-1 released from infected cells inhibited T lymphocyte proliferation in response to interleukin-2 (IL-2). Studies to explore the mechanism of this inhibition suggested that the decrease in proliferation resulted from interactions between HIV-1 and the mouse cells, but did not involve IL-2/IL-2 receptor interactions. We used monoclonal antibodies to demonstrate that the HIV-1 envelope glycoproteins were required for the inhibition of murine T cell proliferation. Anti-gp120 antibodies completely restored proliferation, indicating that the surface protein gp120 was primarily required for the inhibition of proliferation. However, antibodies directed against the transmembrane protein of HIV-1 (gp41) also partially restored lymphocyte proliferation. The functional significance of the HIV-1 envelope protein epitopes recognized by the monoclonal antibodies is discussed.     

Patterns of antibody reactivity to selected human immunodeficiency virus type 1 (HIV-1) gp160 epitopes in infected individuals grouped according to CD4+ cell levels

We examined sera from 160 HIV-infected individuals for antibodies reactive to HIV-1 gp160 epitopes defined by seven synthetic peptides. Seropositive individuals were placed into three groups based upon levels of circulating CD4+ cells. These groups consisted of individuals with (1) more than 400 CD4+ cells, (2) 200–400 CD4+ cells, and (3) fewer than 200 CD4+ cells/mm³. The percentage of sera containing antibodies reactive with two immunodominant gp160 epitopes (a.a. 304–321 and 600–611) was unchanged between groups, regardless of CD4 cell numbers. The percentage of sera containing antibodies reactive with weakly immunogenic gp160 epitopes, such as those defined by peptides 425–448 and 846–860, declined in the groups as CD4 values decreased. Our results suggest that the patterns of antibody reactivity to gp160 epitopes change as CD4 levels decline. A narrowing of the humoral immune response to epitopes on the envelope of HIV-1 appears to occur with disease progression.     

Correlation between gag-specific CD8 T-cell responses, viral load, and CD4 count in HIV-1 infection is dependent on disease status

BACKGROUND: It still remains controversial which kind of relationships exist between HIV-1-specific CD8 T-cell responses and HIV RNA load or CD4 count over the course of the infection. This study was designed to investigate the role of HIV-specific CD8 responses in patients with different disease status. METHODS: Three cohorts of patients were selected according to CD4 count levels: long-term nonprogressors (LTNPs, n = 19), asymptomatic progressors (CD4 counts between 500 and 350 cells/mm(3), n = 14), and progressors (CD4 counts <350 cells/mm(3), n = 23). Six of the LTNPs experiencing a quick loss of CD4 T-cells and another 6 LTNPs with stable CD4 counts were followed up. T-cell responses were studied using interferon (IFN) gamma-ELISpot assay against HIV p24 and 11 pools of HIV-Gag peptides. RESULTS: No significant differences were found in Gag-specific CD8 responses among the 3 cohorts. However, inverse correlations were identified between CD8 responses and CD4 counts in asymptomatic progressors and between CD4 responses and viral loads in progressors. In addition, the sequential dynamics of CD8 responses in 6 LTNPs showed that with a quick loss of CD4 T-cells around the range of 500 to 300 cells/mm(3), more vigorous CD8 responses were induced simultaneously, and plasma viremia was still kept relatively stable. CONCLUSIONS: These data suggest that the relationship between CD8 response and viral load or CD4 count is not universally consistent throughout the entire course of HIV-1 infection. Gag-specific CD8 responses may play differential roles in different stages of HIV-1 infection, and the maintenance of a threshold level of CD4 T-cells may contribute to mediate effective HIV-specific responses in natural control of HIV-1 infection.     

Immune responses during acute and chronic infection with hepatitis C virus

Hepatitis C virus (HCV) induces persistent infection and causes chronic liver disease in most infected patients. Vigorous HCV-specific CD4+ and CD8+ T cell responses against HCV multiple epitopes are necessary for spontaneous viral clearance during the acute phase, but the virus appears to have multiple strategies to evade these defenses. There are relatively few studies on the role of immune responses during the chronic phase of infection. CD4+ T cell responses appear to protect against liver injury and may be important to clearance during interferon and ribavirin based therapy. Classic cytotoxic T cells (CTL) may primarily damage the liver in chronic HCV, but there may be subpopulations of T cells that protect against liver inflammation. Resolution of these outstanding questions is important to the development of a prophylactic vaccine as well as improving therapeutic options for those with chronic infection.     

CTL escape and increased viremia irrespective of HIV-specific CD4+ T-helper responses in two HIV-infected individuals

We investigated whether development of mutations leads to loss of CD8 T-cell recognition in HIV-1 infection and is possibly linked to alterations in HIV-1-specific CD4(+) T-cell responses in 2 HIV-infected individuals. In patient, H434 full genome sequencing of HIV-1 biological clones at early and late time points during disease progression showed development of fixed mutations in 16 predicted HIV-specific CTL epitopes. Loss of T-cell recognition and reactivity against wild-type and mutant epitopes was observed primarily for the HLA-B27-restricted KK10 epitope and HLA-A2-restricted SL9 epitope. Similarly, in patient H671, decreasing numbers of HLA-A3-restricted CD8(+) T cells specific for the wild-type RK9 epitope was observed after CTL escape. Only in patient H434 loss of CTL responses was paralleled by a decrease in HIV-specific IL-2(+) CD4(+) T-helper responses. This suggests that loss of T-cell reactivity may not be directly linked to HIV-specific CD4(+) T-cell responses but that increased viremia after CTL escape may influence CD4(+) T-helper responses.     

HIV infection: first battle decides the war

The traditional view of HIV-1 infection characterized by the slow decline of CD4+ T cells has radically changed in light of recent observations in rhesus macaques and humans of rapid and extensive infection and removal of memory CD4+ T cells in mucosal tissues within the first three weeks of infection. This initial strike to the immune system seems to be the distinguishing feature of HIV-1 pathogenesis and its extent sets the overall course of the ensuing infection. Qualitatively different mechanisms of CD4+ T-cell depletion prevail during the acute, chronic and advanced phases of infection depending on the availability of the target-cell population and competence of the immune system. The elimination of CD4+ T cells in mucosal lymphoid tissues results in the absence of important regulatory and effector functions that these cells normally perform in controlling immune responses to environmental antigens and pathogens. Ablation of acute HIV-1 viremia limits the initial damage to the CD4+ T-cell compartment and helps to establish a state of equilibrium between the replicating virus, the availability of the target-cell population and the immune control characteristic of long-term non-progression.     

DNA-immunization with a V2 deleted HIV-1 envelope elicits protective antibodies in macaques

Rhesus macaques immunized with the HIV-1 SF162DeltaV2 gp140 envelope using the DNA-prime plus protein-boost vaccination methodology, developed HIV envelope-specific T-cell lymphoproliferative responses and potent neutralizing antibodies. To evaluate the protective potential of these antibodies during acute infection, the animals were depleted of their CD8+ T lymphocytes using specific monoclonal antibodies and subsequently challenged intravenously with the pathogenic SHIV(SF162P4) isolate. As compared to non-vaccinated animals (one of which died from AIDS 16 weeks post-exposure) the vaccinated macaques had lower levels of peak viremia, rapidly cleared virus from the periphery and developed delayed seroconversion to SIV core antigens.     

Antibody-dependent cellular cytotoxicity (ADCC) is directed against immunodominant epitopes of the envelope proteins of human immunodeficiency virus 1 (HIV-1).

In this study, epitopes of HIV envelope proteins that are involved in ADCC were identified. Peripheral blood mononuclear cells (PBMC) were obtained from adults with asymptomatic HIV infection or early symptoms of AIDS. These PBMC, which were reported to be "armed" in vivo with HIV-specific antibodies, were used as effector cells in 51Cr release assays. Target cells consisted of CD4 lymphocytes from healthy seronegative donors, coated with the IIIB strain of HIV-1 or with one of seven synthetic peptides. Cytotoxicity was detected against CD4 lymphocytes coated with HIV-1 IIIB or with the peptides env aa 507-518, corresponding to the carboxy-terminus of gp120, and env aa 597-611, corresponding to the region of the cysteine loop of gp41. The magnitude of target cell lysis was directly related to the quantity of peptide used. In contrast, target cells coated with the peptide gag aa 129-135, corresponding to the p17/p24 cleavage region of the gag precursor, were not killed. The same immunodominant regions which were involved in ADCC were recognized in enzyme-linked immunoabsorbent assays (ELISA) by the majority of 107 sera from HIV-infected adults. We conclude that the immunodominant epitopes located at the carboxy-terminus of gp120 and the cysteine loop of gp41 serve as recognition structure for antibodies, capable of mediating ADCC against HIV-infected cells.     

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1).

Antibody mediated and cell mediated immune responses to the envelope glycoproteins gp120 and gp41 of the human immunodeficiency virus (HIV-1) are considered important for protection against infection and for attenuation of disease symptoms after infection. Virus neutralizing antibodies are mostly subtype specific and primarily directed against epitopes on a hypervariable loop from the V3 region of HIV-1 gp120. Such epitopes are recognized by helper and cytotoxic T-cells suggesting that all protective immune responses to HIV-1 are predominantly subtype specific. The extraordinary primary sequence variability of gp120 indicates that a combination of subtype specific components will be required to design a broadly effective protective immunogen against HIV-1. Peptides from hypervariable loops of the V3 region of 21 distinct HIV-1 isolates (clones) were synthesized and used to raise rabbit antisera. The antisera contained high levels of antibodies recognizing the homologous peptides and the parent gp120 sequence. The serological cross-reactivity between the distinct peptides was evaluated and related to amino acid divergence. The corresponding relationship approximated a linear regression with a correlation coefficient r = 0.718. The 21 peptides were combined into a single immunogen which elicited broadly reactive antibodies recognizing all 21 peptides as well as gp120 from the only isolate tested, HIV-1 IIIB. The results suggest the possibility of developing broadly protective HIV-1 immunogens by combining judiciously selected subtype specific peptides derived from envelope glycoproteins of divergent virus isolates.     

In vivo induction of cellular and humoral immune responses by hybrid DNA vectors encoding simian/human immunodeficiency virus/hepatitis B surface antigen virus particles in BALB/c and HLA-A2-transgenic mice

To improve the immunogenicity of epitopes derived from Gag proteins of simian immunodeficiency virus (SIV) and from the envelope (Env) protein of human immunodeficiency virus type 1 (HIV-1), we have designed hybrid DNA vaccines by inserting sequences encoding antigenic domains of SIV and HIV-1 into the hepatitis B virus envelope gene. This gene encodes the hepatitis B surface antigen (HBsAg) capable of spontaneous assembly into virus-like particles that were used here as carrier. Injections of hybrid vectors encoding B-cell epitopes from the gp41 and the gp120 envelope proteins of HIV-1 induced specific humoral responses in BALB/c mice. Furthermore, high frequencies of IFN-gamma-secreting CD8+ T cells specific for various antigenic determinants of SIV-Gag were observed after intramuscular injections of hybrid DNA vectors in BALB/c mice. Genetic immunization of HLA-A2.1-transgenic mice with HIV-Env/HBsAg-encoding DNA generated a strong CTL response and IFN-gamma-secreting CD8+ T lymphocytes specific for HIV-1 envelope-derived peptide. H-2d-restricted HBs-specific T-cell responses dominated over SIV-Gag responses in BALB/c mice whereas HLA-A2-restricted HIV-Env response was enhanced after fusion with HBsAg. These data demonstrate that different B and T-cell epitopes of vaccine-relevant viral antigens can be expressed in vivo as fusion proteins with HBsAg but that the optimal immunogenicity may differ strikingly between individual epitopes.     

High frequencies of antibody responses to CD4 induced epitopes in HIV infected patients started on HAART during acute infection

For many years only two human monoclonal antibodies (HMAbs) recognizing the CD4 induced (CD4i) epitopes of HIV-1 gp120 existed. Although a number of new CD4i HMAbs have been published recently, we have noted that in most attempts to produce HMAbs using EBV transformation a majority of antibodies produced in culture are lost within a few weeks. To determine what kinds of antibodies are made in these cultures we devised a semiquantitative culture to assess the frequency of B cells capable of producing antibodies and a microcompetition assay to determine what kinds of antibodies were made. Our results show that in three patients started on HAART during acute infection the most frequently produced antibodies binding to gp120 were directed against the CD4i epitopes. Our observations suggest that CD4i epitopes are much more immunogenic than had been previously appreciated. It is possible that envelope glycoproteins shed from virions and perhaps complexed with CD4 are responsible for eliciting these antibodies. The preservation of well regulated immune responses in these patients, together with repeated exposure to viral antigens (i.e. env), may explain the presence of larger than usual numbers of env-specific B cells that could be detected in EBV transformed cultures.     

Neutralizing antibodies mechanism of neutralization and protective activity against HIV-1

The role of the humoral immune response in prevention against HIV-1 infection is still incompletely understood. However, neutralizing antibodies to certain epitopes on HIV-1 envelope glycoproteins inhibit HIV-1 infection in vitro and in vivo. Passive administration of these antibodies by themselvesor in combination completely protected hu-PBL-SCID mice or macaques from intravenous, vaginal, as well as maternal-fetal mucosal transmission. All these studies provide direct experimental evidence that neutralizing antibodies are potentenough to prevent HIV infection, and strongly suggest that neutralizing-antibody-based vaccines could provide effective protection against HIV-1, despite the potent action of CTLs. Some neutralizing epitopes have been defined in vitro and in vivo. Unfortunately, none of the neutralizing-antibody-based candidate vaccines has been demonstrated to induce enough protective activity. Weak antigenicity and immunogenicity of neutralizing epitopes on native or recombinant proteins and other factors made it difficult to induce neutralizing-epitope-specific antibody responses in vivo enough to prevent against primary isolates. Recent studies indicated that HIV-1 variations resulted in escape from neutralization or the CTL responses, which may be the principal challenge for HIV-1 prevention. Epitope vaccine as a new strategy activating both arms of the immune system, namely, using the “principal neutralizing epitopes” and the CTL epitopes in combination, should provide new hope for developing an effective vaccine to halt the HIV-1 epidemic.     

Monocyte-mediated T cell suppression by HIV-2 envelope proteins

HIV-2 is associated with an attenuated form of HIV disease. We investigate here the immunosuppressive effects of the HIV-2 envelope protein, gp105. We found that gp105 suppresses activation of T cells through a monocyte-mediated mechanism. Suppression of T cell activation by gp105 depends on contact between monocytes and T cells, but not on CD4+CD25+ T cells. The TLR4 pathway is likely involved, since gp105 activates TLR4 signaling and induces TNF-alpha production by monocytes. Immunosuppression is viewed as the main pathophysiologic consequence of infection by HIV. However, the main immunologic defect caused by HIV, depletion of T cells, requires T cell activation. Our findings are consistent with a new concept that HIV-2 envelope proteins act on monocytes to suppress T cell activation and that this property may contribute to the benign course of HIV-2. We hypothesize that the HIV-2 envelope immunosuppressive properties limit bursts of T cell activation, thus reducing viremia and contributing to the slow rate of disease progression that characterizes HIV-2 disease.     

Multiepitope vaccines intensively increased levels of antibodies recognizing three neutralizing epitopes on human immunodeficiency virus-1 envelope protein

A few neutralizing antibodies against human immunodeficiency virus-1 (HIV-1) envelope proteins have been shown to be highly effective at neutralizing different strains in vitro, and exist at very low levels in the sera of HIV-1-infected individuals. Based on our hypothesis that epitope vaccination may be a novel strategy for inducing high levels of antibodies against HIV-1, we prepared multiepitope vaccines using three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope proteins. The PI [C-G-(ELDKWA-GPGRAFY)2-K] and PII (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) peptides were synthesized and conjugated to a carrier protein, bovine serum albumin (BSA). After vaccination, both the PI-BSA and PII-BSA multiepitope vaccines induced high levels of epitope-specific antibodies to the three neutralizing epitopes (antibody titre: 1 : 12,800-102,400). The recombinant glycoprotein 160 (rgp160) subunit vaccine induced strong antibody responses to rgp160, but only very weak epitope-specific antibody responses to the three epitopes. The epitope-specific antibodies were isolated from rabbit sera by single epitope-peptide-conjugated sepharose columns. A yield of 51 microg of epitope-specific antibodies/ml of serum (mean value) was obtained and identified to recognize these epitopes, while 0.35 microg of protein was isolated from 1 ml of pooled preserum by C-(ELDKWAG)4- or C-(RILAVERYLKD-G)2-K- and C-(GPGRAFY)4-sepharose columns. The levels of these epitope-specific antibodies induced in rabbits were much greater than 1 microg/ml, a level that is considered to confer long-term protection against some viruses. Moreover, these antibodies recognized the neutralizing epitopes on peptides and rgp41. Based on the fact that a very low level of ELDKWA epitope-specific antibodies exist in HIV-1-infected individuals, these results suggesting that synthetic epitope vaccines could induce high levels of multiepitope-specific neutralizing antibodies indicate a new strategy for developing an effective neutralizing antibody-based epitope/peptide vaccine against HIV-1.