HIV-1 recombinant poxvirus vaccine induces cross-protection against HIV-2 challenge in rhesus macaques.

Rhesus macaques were immunized with attenuated vaccinia or canarypox human immunodeficiency virus type 1 (HIV-1) recombinants and boosted with HIV-1 protein subunits formulated in alum. Following challenge with HIV-2SBL6669, three out of eight immunized macaques resisted infection for six months and another exhibited significantly delayed infection, whereas all three naive controls became infected. Immunizations elicited both humoral and cellular immune responses; however, no clear correlates of protection were discerned. Although more extensive studies are now called for, this first demonstration of cross-protection between HIV-1 and -2 suggests that viral variability may not be an insurmountable problem in the design of a global AIDS vaccine.

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Establishment of long-term monocyte suspension cultures from normal human peripheral blood.

The long-term suspension growth of normal, immature myeloid cells from fresh human cord blood was recently reported and required cells separated on supplemented discontinuous Percoll gradients, growth in media containing hydrocortisone and vitamins D3, and gentle, continuous agitation (13). When normal adult bone marrow (six donors) or blood from Epstein-Barr virus (EBV)-seropositive donors (nine donors) was used as a source of fresh human leukocytes, only short-term proliferation of myeloid cells was achieved with the same techniques. However, when leukocytes prepared from EBV seronegative normal adult peripheral blood were used, pure populations of monocytes and macrophages that replicate slowly in liquid suspension culture for greater than 5 mo were repeatedly obtained from three independent donors. These cultures consists of several morphologically distinguishable monocytic cell types, including an approximately 20% adherent macrophage population. The monocytic nature of these cultures was confirmed by cytochemical, immunological, and functional criteria. These monocytes retain a normal chromosome pattern and can be induced to differentiate to phagocytic cells by treatment with tetradecanylphorbal acetate. Eventually, the cultures terminate as nonreplicating mature macrophages. These liquid suspension cultures should be a valuable resource for morphological, biochemical, and functional studies of developing monocyte-macrophages and their interaction with other cell types in normal and various pathological situations.

In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus-6).

We investigated the cellular tropism of human B-lymphotropic virus (HBLV) (also designated Human Herpesvirus-6) in vitro by infecting fresh MN cells from normal human adult peripheral blood, umbilical cord blood, bone marrow, tonsil, and thymus. Cultures from all the sources examined contained infectable cells, as shown by the appearance of characteristic enlarged, round-shaped, short-lived cells expressing HBLV-specific markers. Detailed immunological analysis demonstrated that the vast majority of these cells expressed T cell-associated antigens (i.e., CD7, CD5, CD2, CD4, and to a lesser extent, CD8). The CD3 antigen and the TCR-alpha/beta heterodimer were not detectable on the surface membrane, but were identified within the cytoplasm of HBLV-infected cells, by both immunofluorescence and radioimmunoprecipitation assay. A proportion of the HBLV-infected cell population also expressed the CD15 and class II MHC DR antigens. By means of immunoselection procedures it was possible to show that a consistent proportion of HBLV-infectable cells were contained within the CD3-depleted immature T cell population, while the depletion of CD2+ cells completely abrogated the infectability of the cultures. Northern blot analysis confirmed the T cell origin of HBLV-infected cells, demonstrating the expression of full size TCR-alpha and -beta chain mRNA. In addition to fresh T cells, HBLV was able to infect normal T lymphocytes expanded in vitro with IL-2 for greater than 30 d. These results indicate that HBLV is selectively T cell tropic in the course of the in vitro infection of normal mononuclear cells and may therefore be directly involved in the pathogenesis of T cell related hematological disorders. In particular, in light of the cytopathic effect exerted in vitro on CD4+ T lymphocytes, a possible role of HBLV in immune deficiency conditions should be considered.

Angiogenic properties of Kaposi's sarcoma-derived cells after long-term culture in vitro.

Cells derived from lung biopsies and pleural effusions from AIDS patients with Kaposi's sarcoma (KS) of the lungs were established in long-term culture with the aid of conditioned medium from HTLV-II-transformed T cells (HTLV-II CM). These AIDS-KS cells were similar to the so-called spindle cells in KS lesions and had some of their features. They produced factors that supported their own growth (autocrine) and the growth of other cells (paracrine), including umbilical vein endothelium and fibroblasts. That the AIDS-KS cells also expressed potent angiogenic activity was demonstrated by the chorioallantoic membrane assay and by subcutaneous inoculation of AIDS-KS cells into nude mice, which resulted in the development of angiogenic lesions composed of mouse cells and showing histological features similar to those of human KS lesions. These data suggest that AIDS-associated KS and possibly other types of KS may be initiated by signals that induce the growth of particular cells (spindle cells of lymphatic or vascular origin) and the expression of autocrine and paracrine activities.

Kaposi's sarcoma cells: long-term culture with growth factor from retrovirus-infected CD4+ T cells.

Studies of the biology and pathogenesis of Kaposi's sarcoma (KS) have been hampered by the inability to maintain long-term cultures of KS cells in vitro. In this study AIDS-KS-derived cells with characteristic spindle-like morphology were cultured with a growth factor (or factors) released by CD4+ T lymphocytes infected with human T-lymphotropic virus type I or II (HTLV-I or HTLV-II) or with human immunodeficiency virus type 1 or 2 (HIV-1 or HIV-2). Medium conditioned by HTLV-II-infected, transformed lines of T cells (HTLV-II CM) contained large amounts of this growth activity and also supported the temporary growth of normal vascular endothelial cells, but not fibroblasts. Interleukin-1 and tumor necrosis factor-alpha stimulated the growth of the KS-derived cells, but the growth was only transient and these could be distinguished from that in HTLV-II CM. Other known endothelial cell growth promoting factors, such as acidic and basic fibroblast growth factors and epidermal growth factor, did not support the long-term growth of the AIDS-KS cells. The factor released by CD4+ T cells infected with human retroviruses should prove useful in studies of the pathogenesis of KS.

Inhibition of HIV-1 infection by the beta-chemokine MDC.

CD8(+) T lymphocytes from individuals infected with human immunodeficiency virus-type 1 (HIV-1) secrete a soluble activity that suppresses infection by HIV-1. A protein associated with this activity was purified from the culture supernatant of an immortalized CD8(+) T cell clone and identified as the beta-chemokine macrophage-derived chemokine (MDC). MDC suppressed infection of CD8(+) cell-depleted peripheral blood mononuclear cells by primary non-syncytium-inducing and syncytium-inducing isolates of HIV-1 and the T cell line-adapted isolate HIV-1IIIB. MDC was expressed in activated, but not resting, peripheral blood mononuclear cells and binds a receptor on activated primary T cells. These observations indicate that beta-chemokines are responsible for a major proportion of HIV-1-specific suppressor activity produced by primary T cells.

HTLV-III in saliva of people with AIDS-related complex and healthy homosexual men at risk for AIDS.

Peripheral blood leukocytes and saliva from 20 individuals, including four with the acquired immune deficiency syndrome (AIDS), ten with AIDS-related complex (ARC), and six healthy homosexual males at risk for AIDS, were compared as sources of transmissible human T-cell leukemia (lymphotropic) virus type III (HTLV-III), the virus found to be the etiologic agent of AIDS. All of the AIDS and ARC patients and four of the six healthy homosexuals had evidence of prior exposure to HTLV-III as indicated by seropositivity for antibody to HTLV-III structural proteins. Infectious virus was isolated from the peripheral blood of one of the AIDS patients, four of the ARC patients, and two of the healthy homosexual males, consistent with previous reports. HTLV-III was also isolated from the saliva of four of the ARC patients and four of the healthy homosexuals. Virus was also observed by electron microscopy in material prepared by centrifugation of the saliva of one AIDS patient. Although AIDS does not appear to be transmitted by casual contact, the possibility that HTLV-III can be transmitted by saliva should be considered.

Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders.

A novel human B-lymphotropic virus (HBLV) was isolated from the peripheral blood leukocytes of six individuals: two HTLV-III seropositive patients from the United States (one with AIDS-related lymphoma and one with dermatopathic lymphadenopathy), three HTLV-III seronegative patients from the United States (one with angioimmunoblastic lymphadenopathy, one with cutaneous T-cell lymphoma, and one with immunoblastic lymphoma), and one HTLV-III seronegative patient with acute lymphocytic leukemia from Jamaica. All six isolates were closely related by antigenic analysis, and sera from all six virus-positive patients reacted immunologically with each virus isolate. In contrast, only four sera from 220 randomly selected healthy donors and none from 12 AIDS patients without associated lymphoma were seropositive. The virus selectively infected freshly isolated human B cells and converted them into large, refractile mono- or binucleated cells with nuclear and cytoplasmic inclusion bodies. HBLV is morphologically similar to viruses of the herpesvirus family but is readily distinguishable from the known human and nonhuman primate herpesviruses by host range, in vitro biological effects, and antigenic features.

Genetic variation in HTLV-III/LAV over time in patients with AIDS or at risk for AIDS.

In a study of genetic variation in the AIDS virus, HTLV-III/LAV, sequential virus isolates from persistently infected individuals were examined by Southern blot genomic analysis, molecular cloning, and nucleotide sequencing. Four to six virus isolates were obtained from each of three individuals over a 1-year or 2-year period. Changes were detected throughout the viral genomes and consisted of isolated and clustered nucleotide point mutations as well as short deletions or insertions. Results from genomic restriction mapping and nucleotide sequence comparisons indicated that viruses isolated sequentially had evolved in parallel from a common progenitor virus. The rate of evolution of HTLV-III/LAV was estimated to be at least 10(-3) nucleotide substitutions per site per year for the env gene and 10(-4) for the gag gene, values a millionfold greater than for most DNA genomes. Despite this relatively rapid rate of sequence divergence, virus isolates from any one patient were all much more related to each other than to viruses from other individuals. In view of the substantial heterogeneity among most independent HTLV-III/LAV isolates, the repeated isolation from a given individual of only highly related viruses raises the possibility that some type of interference mechanism may prevent simultaneous infection by more than one major genotypic form of the virus.

Lymphokine production by cultured human T cells transformed by human T-cell leukemia-lymphoma virus-I.

Cell-free conditioned media from human T cells transformed by human T-cell leukemia-lymphoma virus (HTLV-I) were tested for the production of soluble biologically active factors, including several known lymphokines. The cell lines used were established from patients with T-cell leukemia-lymphoma and from human umbilical cord blood and bone marrow leukocytes transformed by HTLV-I in vitro. All of the cell lines liberated constitutively one or more of the 12 biological activities assayed. These included macrophage migration inhibitory factor (MIF), leukocyte migration inhibitory factor (LIF), leukocyte migration enhancing factor (MEF), macrophage activating factor (MAF), differentiation inducing factor (DIF), colony stimulating factor (CSF), eosinophil growth and maturation activity (eos. GMA), fibroblast activating factor (FAF), gamma-interferon and, in rare instances, T-cell growth factor (TCGF). Some cell lines produced interleukin 3 (IL-3), platelet-derived growth factor (PDGF), or B-cell growth factors (BCGF). Such cells should prove useful for the production of lymphokines and as sources of specific messenger RNA's for their genetic cloning.

Cytokines from activated T cells induce normal endothelial cells to acquire the phenotypic and functional features of AIDS-Kaposi's sarcoma spindle cells.

Kaposi's sarcoma (KS) is a proliferative disease of vascular origin particularly frequent in HIV-1-infected homosexual men (AIDS-KS) and characterized by proliferating spindle-shaped cells, angiogenesis, and inflammatory cell infiltration. Previous work has suggested that KS spindle cells are of endothelial cell origin and that chronic immune activation via the release of inflammatory cytokines may cooperate with basic fibroblast growth factor (bFGF) and the HIV-1 Tat protein in the induction and progression of AIDS-KS. Here we show that KS spindle cells have features of activated endothelial cells, and that conditioned media from activated T cells, rich in the same inflammatory cytokines increased in HIV-1-infected individuals, induce normal endothelial cells to acquire the phenotypic and functional features of KS cells. These include (a) acquisition of a similar pattern of cell surface antigen expression; (b) similar proliferative response to bFGF; (c) induction of the responsiveness to the mitogenic effect of extracellular HIV-1 Tat protein that is now able to promote the G1-S transition of endothelial cell cycle; and (d) induction in nude mice of vascular lesions closely resembling early KS as well as the lesions induced by inoculation of KS cells. These results suggest that chronic immune activation, via release of inflammatory cytokines, may play a role in the induction of KS.

Prospective study of cytotoxic T lymphocyte responses to influenza and antibodies to human T lymphotropic virus-III in homosexual men. Selective loss of an influenza-specific, human leukocyte antigen-restricted cytotoxic T lymphocyte response in human T lymphotropic virus-III positive individuals with symptoms of acquired immunodeficiency syndrome and in a patient with acquired immunodeficiency syndrome.

Peripheral blood leukocytes (PBL) from 18 homosexual men who did not have acquired immunodeficiency syndrome (AIDS) and from 9 heterosexual men were repetitively tested for their ability to generate HLA self-restricted cytotoxic T lymphocyte responses to influenza virus (flu-self) over a 2-yr period. The sera of the same donors were tested for antibodies to human T lymphotropic virus-III (HTLV-III). Six of the homosexual and none of the heterosexual donors consistently generated weak cytotoxic T lymphocyte responses to flu-self. Seven of the homosexual and none of the heterosexual donors were seropositive for antibodies to HTLV-III. No obvious correlation was detected between weak flu-self cytotoxic T lymphocyte responses and antibodies to HTLV-III. However, one homosexual donor generated no detectable cytotoxic T lymphocyte activity to flu-self, although he was a strong responder to HLA-alloantigens. This donor had an OKT4:OKT8 ratio of 0.4 and was seropositive for HTLV-III antigens; HTLV-III virus was identified in his PBL; and he developed AIDS during the course of this study. A second donor with lymphadenopathy and who was seropositive for HTLV-III antigens exhibited marginal cytotoxic T lymphocyte activity to flu-self which he subsequently lost. PBL from two patients, one with Kaposi's sarcoma and one with generalized lymphadenopathy, were also tested for cytotoxic T lymphocyte responses to flu-self and to alloantigens. Both donors failed to generate cytotoxic T lymphocyte to flu-self, but generated strong cytotoxic T lymphocyte responses to alloantigens. The selective loss of an HLA-restricted cytotoxic T lymphocyte response without loss of HLA alloantigenic cytotoxic T lymphocyte activity may be an important functional immunologic characteristic in the development of AIDS.

Effect of type C viral expression and cellular karyotype on human B-lymphoblast tumorigenicity in nude mice.

Cultured human hematopoietic cells from several normal and leukemic sources, including those cells initiated after exposure to primate type C retroviruses were tested for their capacity to induce tumors in young athymic BALB/c (nu/nu) mice after sc inoculation. An attempt was made to correlate these results with virus expression and chromosome patterns. Progressively growing tumor formation was observed in 5 of 18 normal diploid B-lymphoblast lines from normal peripheral blood and in one of three diploid B-lymphoblast lines from leukemic donors established after infection with primate type C viruses (gibbon ape leukemia virus or simian sarcoma virus). In contrast, none of eight spontaneously transformed B-lymphoblast lines with normal diploid karyotypes formed progressively growing tumors, although one formed a tumor that remained the same size (0.5 cm) for several months. Progressive tumor formation occurred in four of seven previously established cell lines of different cell types that had abnormal karyotypes. Of the normal diploid B-lymphoblast cultures exposed to type C viruses, 12 were tested for the presence of viral RNA and structural proteins (p12, p30, gp70), and this information was correlated with tumorigenicity. Four of the six cultures expressing viral RNA or proteins were tumorigenic, whereas only one of six cultures that did not express virus information was positive. The results of this study suggest that expression of type C viral RNA and proteins by human B-lymphoblasts increases their tumorigenicity in nude mice. It is also apparent that caution must be used in attempts to correlate cell tumorigenicity and chromosome abnormalities in nude mice.

Advances in the isolation of HTLV-III from patients with AIDS and AIDS-related complex and from donors at risk.

During the last 2 yr more than 100 independent isolates of human T-cell leukemia virus type III have been obtained in this laboratory. Most isolates were from peripheral blood T-lymphocytes established in cell culture from acquired immunodeficiency syndrome (AIDS) and acquired immunodeficiency syndrome-related complex (ARC) patients and healthy donors at risk for AIDS. Several were also obtained from leukocytes from bone marrow, lymph node, and brain tissue and from body excretions, e.g., saliva and semen. In addition HTLV-III was found in cell-free plasma. The incidence (number of isolates per number of patients or donors tested) of virus isolation was approximately 80% for ARC patients, approximately 50% for AIDS patients, and approximately 30% for healthy individuals at risk for AIDS. Inclusion of hydrocortisone in cell culture medium greatly facilitated the isolation of virus from primary leukocytes from AIDS/ARC patients and also promoted its transmission to fresh leukocytes in vitro. Biological analysis of cells from infected patients or donors, as well as from normal peripheral blood mononuclear cells exposed to virus in vitro, demonstrated that OKT4/leu3a+ T-lymphocytes were preferentially infected and were subjected to a characteristic cytopathic effect. In addition to the well-defined individuals at risk for AIDS, heterosexual transmission of HTLV-III with its subsequent pathological manifestations was found. Virus was isolated from males with heterosexual promiscuity as their only recognized risk factor and from the spouses of these and other AIDS and ARC patients.

Restricted expression of retrovirus nucleic acids and proteins in primate type C virus (gibbon ape leukemia virus-simian sarcoma virus)-initiated human B-lymphoblast cultures.

Fresh human B-lymphoblasts established in culture following exposure of adult peripheral blood leukocytes to type C retroviruses of the simian sarcoma virus/simian sarcoma-associated virus-gibbon ape leukemia virus group were analyzed in detail for the presence of the infecting virus. Viral expression ranged from production of low levels of intact virus in a few cultures to the presence of viral RNA and protein in the absence of detectable of levels of complete virus in the majority of the cultures. In situ molecular hybridization assays using 3H-labeled complementary DNA and indirect immunofluorescence assays using antibody to purified viral protein indicated that the expression of viral RNA and proteins are preferentially expressed in only a fraction of the cells in some cultures. If expression of the infecting viral sequences is necessary for the sustained growth of these cells, then those cells detectably synthesizing viral RNA and proteins may be influencing the growth of the remaining virus-negative cells. The lack of virus production in cultures synthesizing viral RNA and protein indicate that these human B-lymphocytes restrict the life cycle of these viruses at some step(s) after transcription of viral RNA or translation of viral protein.

Cross-protection in NYVAC-HIV-1-immunized/HIV-2-challenged but not in NYVAC-HIV-2-immunized/SHIV-challenged rhesus macaques.

OBJECTIVES: Immunization with attenuated poxvirus-HIV-1 recombinants followed by protein boosting had protected four of eight rhesus macaques from HIV-2SBL6669 challenge. The present study was designed to confirm this result and to conduct the reciprocal cross-protection experiment. METHODS: Twenty-four macaques were primed with NYVAC (a genetically attenuated Copenhagen vaccinia strain) recombinants with HIV-1 and HIV-2 env and gag-pol or NYVAC vector alone and boosted with homologous, oligomeric gp160 proteins or adjuvant only. Binding and neutralizing antibodies, cytotoxic T-lymphocytes (CTL) and CD8 T cell antiviral activity (CD8AA) were evaluated. One half of each immunization and control group were intravenously challenged with SHIV(HXB2) the other half was challenged with HIV-2SBL6669,. Protective outcome was assessed by monitoring virus isolation, proviral DNA and plasma viral RNA. RESULTS: Both immunization groups developed homologous binding antibodies; however, homologous neutralizing antibodies were only observed in NYVAC-HIV-2-immunized macaques. While no cross-reactive neutralizing antibodies were detected, both immunization groups displayed cross-reactive CTL. Significant CD8AA was observed for only one NYVAC-HIV-2-immunized macaque. Virological assessments verified that both NYVAC-HIV-1 and NYVAC-HIV-2 immunization significantly reduced viral burdens and partially protected against HIV-2 challenge, although cross-protection was not at the level that had been previously reported. Humoral antibody and/or CTL and CD8AA were associated with protection against homologous HIV-2 challenge, while cellular immune responses seemed more important for cross-protection. No significant protection was observed in the SHIV-challenged macaques, although NYVAC-HIV-1 immunization resulted in significantly lower viral burdens compared with controls. CONCLUSIONS: Further delineation of cross-reactive mechanisms may aid in the development of a broadly protective vaccine.

Multiple immunizations with attenuated poxvirus HIV type 2 recombinants and subunit boosts required for protection of rhesus macaques.

Vaccine protocols involving multiple immunizations with molecularly attenuated vaccinia virus (NYVAC) or naturally attenuated canarypox virus (ALVAC) HIV-2 recombinants and subunit boosts have conferred longlasting protection against HIV-2 infection of macaques. Similar complex protocols using HIV-1 NYVAC and ALVAC recombinants and subunit boosts have provided cross-protection against HIV-2 challenge. Here a simplified three-immunization regimen over 24 weeks was tested in 18 juvenile rhesus macaques. Twelve macaques were immunized twice with NYVAC or ALVAC recombinants carrying HIV-2 env, gag, and pol genes. Subsequently, macaques in groups of three received either an additional recombinant immunization or an HIV-2 gp160 boost. Six control macaques received three immunizations of NYVAC or ALVAC vector alone and additionally alum at the third immunization. Macaques primed with ALVAC recombinant exhibited sporadic T cell proliferative activity, and all but one failed to develop neutralizing antibodies. In contrast, macaques primed with NYVAC recombinants had no T cell proliferative activity but exhibited neutralizing antibody titers (highest in the three recombinant group) that declined by the time of challenge. None of the macaques exhibited significant cytotoxic T lymphocyte activity. Following challenge at 32 weeks with HIV-2SBL6669 all macaques became infected. Thus, the three-immunization regimen is not sufficient to confer protective immunity in the HIV-2 rhesus macaque model. However, delayed infection in macaques immunized with the NYVAC-HIV-2 recombinant may have been associated with the development of memory B cells capable of providing a neutralizing antibody response on challenge.

A conformational C4 peptide polymer vaccine coupled with live recombinant vector priming is immunogenic but does not protect against rectal SIV challenge.

The conserved, immunogenic CD4 binding site on the viral envelope is an attractive HIV or SIV vaccine candidate. Polymerization of an 18 amino acid segment derived from the C4 domain of SIV gp120 produced a peptide polymer or "peptomer," having an alpha-helical conformation possibly mimicking a proposed structure of the C4 domain in the unbound native protein. The SIV peptomer and native gp120 were compared as subunit boosts following two adenovirus type 5 host range (Ad5hr)-SIVenv recombinant priming immunizations. Both vaccine regimens successfully elicited SIV-specific CTL responses in five of six immunized macaques. Peptomer-boosted macaques exhibited significantly higher envelope-specific T cell proliferative responses than either the gp120-boosted macaques or controls. Peptomer immunization also elicited peptomer and SIV gp120-specific binding antibodies, but only native gp120 boosting elicited SIV neutralizing antibodies. Upon intrarectal challenge with SIVmac32H, all nine macaques became infected. The solely envelope-based vaccine conferred no protection. However, changing the boosting immunogen to the C4 peptomer did not improve protective efficacy in spite of its elicitation of humoral and cellular immune responses, including robust T-helper activity. In spite of the peptomer's strong immunogenicity and potential for induction of broadly protective immune responses, it was not effective as a subunit vaccine.