Characterization of CD4 and CD8 T cell responses in MuSK myasthenia gravis.

Muscle specific tyrosine kinase myasthenia gravis (MuSK MG) is a form of autoimmune MG that predominantly affects women and has unique clinical features, including prominent bulbar weakness, muscle atrophy, and excellent response to therapeutic plasma exchange. Patients with MuSK MG have predominantly IgG4 autoantibodies directed against MuSK on the postsynaptic muscle membrane. Lymphocyte functionality has not been reported in this condition. The goal of this study was to characterize T cell responses in patients with MuSK MG. Intracellular production of IFN-gamma, TNF-alpha, IL-2, IL-17, and IL-21 by CD4+ and CD8+ T cells was measured by polychromatic flow cytometry in peripheral blood samples from 11 Musk MG patients and 10 healthy controls. Only one MuSK MG patient was not receiving immunosuppressive therapy. Regulatory T cells (Treg) were also included in our analysis to determine if changes in T cell function were due to altered Treg frequencies. CD8+ T cells from MuSK MG patients had higher frequencies of polyfunctional responses than controls, and CD4+ T cells had higher IL-2, TNF-alpha, and IL-17. MuSK MG patients had a higher percentage of CD4+ T cells producing combinations of IFN-gamma/IL-2/TNF-gamma, TNF-alpha/IL-2, and IFN-gamma/TNF-alpha. Interestingly, Treg numbers and CD39 expression were not different from control values. MuSK MG patients had increased frequencies of Th1 and Th17 cytokines and were primed for polyfunctional proinflammatory responses that cannot be explained by a defect in CD39 expression or Treg number.

Polyfunctional cytomegalovirus-specific immunity in lung transplant recipients receiving valganciclovir prophylaxis.

Cytomegalovirus (CMV) is a common opportunistic infection after lung transplant. Despite effective antiviral medications to treat CMV, invasive CMV disease contributes to lung allograft dysfunction and worse survival. Efforts to prevent CMV have led to the use of valganciclovir prophylaxis for increasingly longer periods after transplant. A pivotal concern with long-term antiviral prophylaxis is that it may prevent or delay the development of CMV-specific immunity and increase the subsequent risk of late onset disease. To address this issue, we conducted a pilot study to determine if CMV-specific immunity was detectable in lung transplant recipients at risk for CMV while on antiviral prophylaxis. Utilizing polychromatic flow cytometry panels, CMV-specific immunity was determined by peripheral blood CD4 and CD8 T cell expression of cytokines in response to the HLA restricted CMV peptides pp65 and IE-1. We determined CMV seropositive lung transplant recipients on valganciclovir for a median of 6 months from transplant have a detectable polyfunctional CMV-specific T cell response which is comparable to seropositive recipients not on antiviral medications and to healthy seropositive nontransplant controls. Thus, valganciclovir prophylaxis does not appear to impair the development of CMV-specific immunity in lung transplantation.

The tumor dormant state. Quantitation of L5178Y cells and host immune responses during the establishment and course of dormancy in syngeneic DBA/2 mice.

Subcutaneous implantation of DBA/2-derived L5178Y cells into DBA/2 mice followed 10 d later by nodule excision protected 100% of mice from the rapid outgrowth of an intraperitoneal challenge of L5178Y cells given 7 d postexcision. Challenged mice remained clinically normal for 48--250 d before onset of an ultimately fatal tumor outgrowth. The numbers of L5178Y cells in the peritoneal cavity increased logarithmically for 4 d after challenge and then declined to low but detectable levels which persisted throughout the clinically normal period. Cells active in 18-h in vitro cytolytic assays against 51Cr-labeled L5178Y target cells were found in the peritoneal cavity. The effector cells were determined to be Thy1.2 positive. Their activity was tumor specific and reached peak levels 4 d after tumor challenge and then gradually declined to undectable levels during the following 70 d. Tumor emergence occurred most frequently during the period when CMC activity was no longer demonstrable in the remaining clinically normal mice. A transient peak of low level cytophilic antitumor antibody was detected about 30 d after tumor cell challenge. The temporal associations between the numbers of tumor cells and the levels of cell-mediated lysis against L5178Y cells indicate the importance of the cell-mediated cytolysis response in limiting initial tumor outgrowth and suggest its role as one of the factors responsible for long-term tumor suppression during tumor dormancy.

The tumor dormant state. Comparison of L5178Y cells used to establish dormancy with those that emerge after its termination.

The tumor dormant state established in L5178Y immunized and challenged mice is characterized by a prolonged period of clinical normalcy followed by rapid tumor outgrowth. The tumor cells which emerged after termination of the tumor dormant state had abnormal marker chromosomes identical to those in the L5178Y cells used in the original challenge inoculum, indicating that the emergent tumor cells were progeny of the challenge inoculum. Original and emergent L5178Y cells had equivalent in vivo growth rates, when inoculated into normal DBA/2 mice. The emergent L5178Y cells were less susceptible than original cells to in vitro lysis by tumor dormant PC. Original and emergent L5178Y cells expressed common tumor-associated target antigens for cytolytic effector cells. Both modulation and masking of these target antigens were ruled out as mechanisms for decreased susceptibility to cell-mediated cytolysis. Immunofluorescence revealed heterogeneity in tumor-associated antigen expression within both original and emergent cell populations, with a decreased intensity of staining in the emergent population. Both populations were equally susceptible to lysis by alloimmune cells, alloantiserum, and anti-Thy 1.2 serum, but emergent cells were less susceptible to lysis by serum directed against L5178Y TAA. Quantitative absorption revealed that the emergent L5178Y cells expressed eightfold less serologically detectable TAA than the original cells. These findings indicate that the host immune response developing during establishment of the tumor dormant state selects a stable tumor cell subpopulation which expresses decreased amounts of surface tumor-associated target antigens.

Phenotypic variation in the response to the human immunodeficiency virus among derivatives of the CEM T and WIL-2 B cell lines.

Derivatives of the CEM T and WIL-2 B cell lines showed striking diversity in their responses to the HTLV-IIIB strain of the human immunodeficiency virus (HIV). Several stable phenotypic patterns could be defined, based on whether cells were permissive (P+, P-) for virus production, were sensitive or insensitive to cytopathic effects after infection by free virus (C+, C-), and whether they underwent fusion on contact with virus-infected cells (F+, F-). Although expression of CD4 was essential for infection by HTLV-IIIB, very low levels were sufficient for productive infection of WIL-2 derivatives. Conversely, some CEM T cell lines that expressed ample CD4, and which were able to bind virus gp120 and undergo fusion, did not support productive infection by free virus. One nonpermissive, CD4+ derivative of CEM could bind gp120 but failed to undergo fusion, suggesting an alteration in some membrane protein other than CD4 that is essential for virus entry and HIV-induced cell fusion. The AA2 derivative of the WIL-2 cell line is also described, which is remarkably permissive for HIV replication and exquisitely sensitive to virus cytopathic effect. The panel of related cell lines with different host-virus phenotypes could be useful for more precisely defining steps in the infectious cycle of HIV, and for identifying host cell genes and gene products that determine the outcome of HIV infection.

HTLV-III/LAV-neutralizing antibodies to an E. coli-produced fragment of the virus envelope.

Immunization with either an Escherichia coli recombinant segment of the human T-cell lymphotropic virus (HTLV-III/LAV) envelope protein (gp 120) or with deglycosylated gp 120 envelope protein produced antibodies that neutralize HTLV-III/LAV infection in vitro. Virus neutralization titers of these antisera were equivalent to those obtained with purified native gp120 as immunogen. This localizes at least one class of neutralizing epitopes to the carboxyl-terminal half of the molecule. In addition, native gp120 prevented HTLV-III/LAV--mediated cell fusion, whereas the recombinant gp120 fragment did not. This shows that although glycosylation is not required for induction of neutralizing antibodies, it may be important for interaction with CD4, the virus receptor. A segment of the HTLV-III/LAV envelope produced in E. coli may be an important ingredient of a vaccine for acquired immune deficiency syndrome.

HIV infection--induced posttranslational modification of T cell signaling molecules associated with disease progression.

In attempt to elucidate the mechanism of the HIV infection induced T cell unresponsiveness, we studied signal-transducing molecules proximal to the T cell receptor (TCR) in T lymphocytes of HIV-infected individuals. Total amounts of protein tyrosine kinases (PTKs) Lck, Fyn, and ZAP-70 and the zeta chain of the TCR were found significantly decreased in T cells of symptomatic/AIDS patients as well as in T cells of individuals in acute and early asymptomatic stages of HIV infection. Unexpectedly, the detection of Lck, Fyn, and ZAP-70 was reversed after the treatment of cell lysates with dithiothreitol. This suggests that PTKs Lck, Fyn, and ZAP-70 were modified by a mechanism altering the status of sulfhydryl groups. Moreover, this mechanism seems to affect selectively T cells of HIV infected patients since B cell PTKs Syk and Lyn were detected structurally and functionally intact. Interestingly, similar alterations of signaling molecules were not detected in T cells of HIV-infected long-term asymptomatic individuals. Modification of T cell PTKs may thus underlie the HIV-induced impairment of lymphocyte function and may potentially predict disease progression.

Analysis of the adult thymus in reconstitution of T lymphocytes in HIV-1 infection.

A key question in understanding the status of the immune system in HIV-1 infection is whether the adult thymus contributes to reconstitution of peripheral T lymphocytes. We analyzed the thymus in adult patients who died of HIV-1 infection. In addition, we studied the clinical course of HIV-1 infection in three patients thymectomized for myasthenia gravis and determined the effect of antiretroviral therapy on CD4(+) T cells. We found that five of seven patients had thymus tissue at autopsy and that all thymuses identified had inflammatory infiltrates surrounding lymphodepleted thymic epithelium. Two of seven patients also had areas of thymopoiesis; one of these patients had peripheral blood CD4(+) T-cell levels of <50/mm3 for 51 months prior to death. Of three thymectomized patients, one rapidly progressed to AIDS, one progressed to AIDS over seven years (normal progressor), whereas the third remains asymptomatic at least seven years after seroconversion. Both latter patients had rises in peripheral blood CD4(+) T cells after antiretroviral therapy. Most patients who died of complications of HIV-1 infection did not have functional thymus tissue, and when present, thymopoiesis did not prevent prolonged lymphopenia. Thymectomy before HIV-1 infection did not preclude either peripheral CD4(+) T-cell rises or clinical responses after antiretroviral therapy.

Excellent safety and tolerability of the human immunodeficiency virus type 1 pGA2/JS2 plasmid DNA priming vector vaccine in HIV type 1 uninfected adults.

A vaccine consisting of DNA priming followed by recombinant modified vaccinia Ankara (rMVA) boosting has achieved long-term control of a pathogenic challenge with a chimera of simian and human immunodeficiency viruses (SHIV-89.6P) in rhesus macaques. Based on these results, clade B HIV-1 DNA and rMVA immunogens have been developed for trials in humans. We conducted a first-time in humans phase I safety trial using the pGA2/JS2 (JS2) HIV-1 DNA priming vector expressing Gag, Pol, Env, Tat, Rev, and Vpu. Thirty HIV-uninfected adults were vaccinated with 0.3 or 3 mg of JS2 DNA, or a saline placebo, by intramuscular injection at months 0 and 2. Both doses of DNA were safe and well-tolerated with no differences between the control, 0.3 mg, or 3 mg groups (n = 6, 12, and 12, respectively) through 12 months of postvaccination follow- up. A chromium-release assay using fresh peripheral blood mononuclear cells (PBMCs) and a validated IFN-gamma ELISpot assay with frozen PBMCs failed to detect CD4(+) or CD8(+) HIV-1-specific T cell responses. HIV-specific neutralizing antibodies were also not detected. The vaccine is being further developed as a priming vector for a combined DNA plus rMVA prime/boost HIV vaccination regimen.

Immunologic control of a retrovirus-associated murine adenocarcinoma. VIII. Corynebacterium parvum-activated natural killer cells as potent antibody-dependent cell-mediated cytotoxicity effectors.

The antibody-dependent lytic activity of Corynebacterium parvum-induced peritoneal exudate cells was examined in vitro by utilizing AD755a tumor targets and a homologous anti-AD755a hyperimmune serum. Maximum antibody-dependent cell-mediated cytolysis (ADCC) of tumor targets was achieved within 4 hours of incubation. ADCC activity was found primarily in the plastic nonadherent cell population and was greatly enriched following removal of phagocytic cells by carbonyl iron. Phenotypically, the cells active in short-term ADCC were Qa-5+, ASGM-1+, Thy 1.2+, and NK 1.1+ and were unaffected by treatment with Lyt 1.2, Lyt 2.2, MAC-1, or I-Ab antibodies plus complement. Cells active in antibody-independent lysis of AD755a targets were phenotypically identical to antibody-dependent effectors. Although indicative of a natural killer (NK) cell phenotype, C. parvum-induced effectors differed from "spontaneous" splenic NK cells in their relative sensitivity to anti-Thy 1.2 as well as to anti-NK 1.1 treatment. Unlike the IgG2a-dependent lysis of AD755a-derived cells by inflammatory macrophages, all IgG isotypes of antiAD755a serum were equally effective in ADCC mediated by C. parvum NK cells. Finally, treatment of C. parvum-inoculated animals with anti-ASGM-1 serum eliminated in vitro NK activity and abrogated the in vivo therapeutic effects of hyperimmune serum. These findings, together with other correlations detailed herein, strongly suggested that C. parvum-activated NK cells appeared to represent a unique subset of NK cells that can serve as potent effectors in the antibody-dependent killing of AD755a tumor cells.

Immunotherapy of a murine leukemia virus-infected, chemically induced murine sarcoma with antiviral antibodies.

Many murine tumor models associated with murine leukemia virus(es) (MuLV) have been successfully treated by passive administration of antiviral antibodies. There is a large body of virus-negative tumors, however, which are lowly antigenic and thus refractory to such approaches. Therefore, an investigation was done for determination of whether such tumors could be rendered susceptible to passive serum therapy by introduction of MuLV antigens onto the tumor cell surface. For this purpose a 3-methylcholanthrene-induced fibro-sarcoma from inbred C57BL/6J mice was chosen. Following infection of the tumor in vitro with Friend murine leukemia virus (F-MuLV), the tumor was found to be susceptible to treatment with a high-titered heterologous anti-F-MuLV gp71 antiserum. The specificity of the treatment was determined by conduction of the therapy on the uninfected parental tumor, in which case there was no effect. In addition, therapy could be initiated at time points when demonstrable tumors were present and successfully treated animals were resistant to rechallenge with the infected tumor. Thus conversion of a lowly antigenic virus-negative tumor to an MuLV-positive antigenic tumor rendered such growths susceptible to immunologic management.

Immunologic control of a retrovirus-associated murine adenocarcinoma. VI. Augmentation of antibody-dependent killing following quantitative and qualitative changes in host peritoneal cells.

Attempts were made to augment the antibody-dependent killing of the ascitic AD755a tumor in vivo to protect C57BL/6J mice against the outgrowth of larger tumor burdens. The lethal dose for this tumor is less than 100 cells, and antibodies contained in a hyperimmune antitumor serum (HIS) were found to suppress the outgrowth of a maximum of about 5 X 10(5) cells. Thioglycollate injected ip increased the number of peritoneal macrophages, potential effectors for antibody-dependent cell-mediated cytotoxicity (ADCC), by tenfold to fortyfold and raised the maximum treatable tumor challenge (MTTC) to about 4 X 10(6) cells. By comparison, ip injection of Corynebacterium parvum increased the total peritoneal cell population by only twofold but raised the MTTC to about 20 X 10(6) cells. Neither agent alone had an effect on long-term survival, even at very low tumor inocula (1 X 10(3) cells). The protective HIS is known to contain tumor-binding antibodies in each of the IgG1, IgG2A, and IgG2B isotype fractions. Although the IgG2A fraction is far superior in vivo in the suppression of tumor outgrowth, the IgG2A fraction was also found to be most effective in combination with thioglycollate treatment in agreement with the observed preference of thioglycollate-elicited macrophages for this isotype in in vitro killing assays. In contrast following C. parvum treatment, all three isoptype fractions were equally suppressive to tumor outgrowth. A second major change following C. parvum treatment was that tumor cells precoated in vitro with antibodies were effectively eliminated in vivo. The same antibody-coated cells administered to thioglycollate-treated or unmanipulated animals were uniformly lethal even at much lower tumor doses. Taken together these results suggested a major qualitative change in the antibody-dependent tumor-killing process following C. parvum treatment. This change was most likely due to the C. parvum activation of highly lytic effector cells for ADCC, the identity of which was examined in an accompanying manuscript.

Immunologic control of a retrovirus-associated murine adenocarcinoma. VII. Tumor cell destruction by macrophages and IgG2A.

The mechanism by which IgG2A from a syngeneic antitumor hyperimmune serum mediates destruction of target cells in the presence of thioglycollate-elicited peritoneal macrophages was investigated by using an in vitro assay system. Labeled tumor cells were found to exhibit a biphasic pattern of binding to the effector cells; this binding pattern was dependent on the presence of specific antibody. The initial binding phase produced no apparent changes in the target cell population. Target cells coated with specific antibody exhibited a similar early binding phase, but excess free antibody was required for the subsequent binding phase and its associated release of radiolabel and cell destruction. Several features of this process observed distinguished it from more conventional forms of antibody-dependent cell-mediated cytoxicity. These included 1) the preference for antibodies of the IgG2A isotype, 2) the association of cell destruction with the release of nuclear but not cytoplasmic label, and 3) the requirement of excess free antibody for target cell killing.

Murine in vitro antigenic modification of tumor cells: effect on susceptibility to natural cell-mediated cytotoxicity.

A 3-methylcholanthrene [(MCA) CAS: 56-49-5]-induced fibrosarcoma cell line and its Friend murine leukemia virus-infected counterpart were assessed for their susceptibility to lysis by so-called "natural" effector cells in a series of 51Cr release assays. Detailed functional and phenotypic analysis of lytic effector cell populations from normal C57BL/6 mouse spleens revealed an identity most closely associated with natural cytotoxic cells. Neither tumor cell line was found to be sensitive to natural killer-mediated lysis. Additionally, virus infection of the MCA-induced fibrosarcoma cell line did not affect susceptibility to natural cell-mediated cytotoxicity.

In vivo antigenic modification of tumor cells. III. Metastatic thymic lymphoma specifically infected by thymotropic retrovirus.

Tissue distribution of radiation leukemia virus (RadLV) was examined after its inoculation into normal C57BL/6J (B6) mice, B6 mice bearing a transplantable, non-virus-producing thymic lymphoma (RL12-NP), and B6 mice bearing a transplanted non-virus producing, Harvey murine sarcoma virus-transformed fibrosarcoma. Virus expression was determined by competition radioimmunoassay for murine leukemia virus (MuLV) p30 (predominant group-reactive antigen of MuLV) and for RadLV p12 (a highly type-specific MuLV polypeptide) and by membrane immunofluorescence for cell surface gp71 (predominant envelope glycoprotein of MuLV). Normal adult B6 mice were given three sequential iv injections of RadLV and were examined several times up to 200 days later for the appearance of neoplastic disease or expression of virion antigens. No clinical abnormalities were noted, and animals remained healthy for greater than 200 days. Significant levels of MuLV p30 and RadLV p12 were detected only in the thymuses. Organs and tumors from RL12-NP-inoculated animals contained low or nondetectable levels of virion antigens. Inoculation of mice with RL12-Rad, a cell line derived by in vitro infection of RL12-NP cells with RadLV, produced widespread, discrete metastatic tumors and infiltrated the lymphoid organs of B6 mice in a pattern identical to that observed after administration of RL12-NP cells. Lymphoid organs of RL12-Rad-inoculated animals expressed variable levels of virion antigens reflecting differences in the extent of tumor cell infiltration as opposed to virus spread from tumor to host cells. Administration of infectious RadLV systemically into RL12-NP tumor-bearing animals converted these tumors to viron antigen expressors with levels in superinfected tumors equivalent to those found in RL12-Rad-induced tumors. Infection was highly selective, and host tissues were minimally contaminated by the inoculated virus. Part of this selectivity was explained by the thymotropic property of RadLV. A rapidly dividing murine fibrosarcoma was not infected by RadLV, but this same non-virus-expressing tumor could be infected by common fibrotropic MuLV isolates.

Cross-reacting antigens on L5178Y cells which serve as targets for cytotoxic T-lymphocyte lysis during establishment of the tumor dormant state.

We have attempted to identify the antigen on L5178Y cells that is the target for peritoneal cytotoxic T-lymphocytes (CTL) generated in L5178Y-O (original) immunized and challenged DBA/2 mice during establishment of the L5178Y cell tumor dormant state. These CTL exhibited in vitro lytic activity against methylcholanthrene-induced L5178Y and P815Y cells as well as Gross virus-induced BALB/c cells but not against a variety of other H-2d-tumor cell lines. The pattern of susceptibility to cell-mediated immune cytolysis was identical to the pattern of AB-dependent complement-mediated cytolysis produced by a rabbit anti-L5178Y antiserum. Quantitative expression of surface H-2d determinants was not a limiting factor in tumor cell lysis by CTL. The degree of CTL lysis of the susceptible cell lines was directly related to the amount of tumor-associated antigen expressed on the cell surface. The pattern of in vitro susceptibility to CTL lysis correlated well with in vivo transplantation resistance. The L5178Y cell antigen target for both CTL and Ra-anti-L5178Y serum lysis is likely to be either an endogenous AKR ecotropic viral glycoprotein with a molecular weight of 71,000 or one of two cell surface determinants, at Mr 85,000 and 135,000. These higher-molecular-weight antigens are neither endogenous AKR ecotropic viral-induced nor murine leukemia virus group-specific precursor structural proteins but may be transformation antigens shared by the susceptible tumor cell lines.

MAGE-1-specific precursor cytotoxic T-lymphocytes present among tumor-infiltrating lymphocytes from a patient with breast cancer: characterization and antigen-specific activation.

A potential target for development of tumor-specific immunotherapeutic strategies is the MAGE-1 gene. We have utilized a recently developed recombinant canarypox (ALVAC) virus vector containing the MAGE-1 gene (vCP235) to activate CTLs from a breast cancer patient bearing a MAGE-1+ tumor. Tumor-infiltrating lymphocytes (TILs) obtained from the tumor of a patient were stimulated in vitro with irradiated autologous peripheral blood mononuclear cells acutely infected with the vCP235 construct. These TILs preferentially expanded approximately 6-fold over a 16-day culture period and specifically recognized an allogeneic transformed B-cell line acutely infected with a vaccinia-MAGE-1 recombinant targeting vector (vP1188) in the context of HLA-A2 and/or B7. TCR V beta analysis of in vitro expanded T cells by a quantitative multiprobe RNase protection assay revealed preferential expansion of TCR V beta 6.3 and V beta 6.4. In addition, homologous T-cell receptor beta CDR3 joining sequences were found in the in vitro stimulated cultures. These results suggest that tumor antigen-specific, MHC-restricted CTLs may be derived from precursor CTLs present in TILs obtained from patients with MAGE-1+ tumors by in vitro stimulation with recombinant avipox MAGE-1 virus-infected autologous cells. Collectively, these findings provide a rationale for tumor-associated antigen-based immunization as a means of activating precursor CTLs residing in patients with tumors expressing defined tumor-associated antigens such as MAGE-1.

Safety and immunogenicity of an HLA-based HIV envelope polyvalent synthetic peptide immunogen. DATRI 010 Study Group. Division of AIDS Treatment Research Initiative.

OBJECTIVE: To evaluate the safety and immunogenicity of a polyvalent (PV) HIV envelope synthetic peptide immunogen, C4-V3. The immunogen comprised four peptides containing T-helper epitopes from the fourth constant region (C4) of gp120 of HIV-1MN, and T-helper, cytotoxic T-lymphocyte HLA-B7-restricted, and B-cell neutralizing epitopes from the gp120 third variable region (V3) of four clade B HIV-1 isolates, HIV-1MN, HIV-1RF, HIV-1EV91, and HIV-1Can0A. DESIGN: A pilot, Phase I controlled trial [Division of AIDS Treatment Research Initiative (DATRI) 010] conducted at a single center. METHODS: Ten HIV-infected, HLA-B7-positive patients with CD4 cells > 500 x 10(6)/l were enrolled. Eight patients received the C4-V3 PV immunogen emulsified in incomplete Freund's adjuvant in five intramuscular injections over 24 weeks, and two controls received incomplete Freund's adjuvant alone. All subjects were followed for 52 weeks. RESULTS: Four out of eight C4-V3 PV recipients generated at least fourfold rise in serum antibody titers to at least three immunogen peptides in contrast to none of the control subjects. Four out of eight C4-V3 PV recipients and none of the controls had an at least fourfold rise in neutralizing antibodies to either HIV-1MN, HIV-1RF, or HIV-1(4489-5) laboratory-adapted HIV isolates. 3H-Thymidine incorporation assays of peripheral blood mononuclear cells increased at least fivefold over the baseline stimulation index to at least one of the immunogen peptides in two consecutive post-immunization timepoints in five out of eight C4-V3 PV recipients versus none of the controls. CD4 cell counts and plasma HIV RNA levels did not change in patients who received either C4-V3 PV or adjuvant alone. Adverse events consisted primarily of grade 1 injection site reactions in six subjects (four C4-V3 recipients, two controls). CONCLUSIONS: C4-V3 PV synthetic peptides demonstrated both immunogenicity and safety in HIV-infected patients.

Induction of immune responses to HIV-1 by canarypox virus (ALVAC) HIV-1 and gp120 SF-2 recombinant vaccines in uninfected volunteers. NIAID AIDS Vaccine Evaluation Group.

OBJECTIVE: To determine the ability of live attenuated canarypox virus expressing HIV antigens to induce CD8+ cytotoxic T-cell responses and to prime for neutralizing antibody responses to boosting with purified recombinant gp120 subunit vaccine. DESIGN: A prospective, double-blind, randomized, immunogenicity and safety study was conducted in healthy adults at low risk for acquiring HIV infection and who were seronegative for HIV. METHODS: CD8+ cytotoxic T-cells directed against Env or Gag expressing target cells were measured after live recombinant canarypox-HIV-1 vaccine priming (vaccine given at days 0, 7, 14 and 21). Neutralizing antibodies were measured after subunit boosting (vaccine given at days 28 and 84). RESULTS: CD8+ CTL were induced in 64% of volunteers by the live recombinant canarypox-HIV-1 vaccine. All volunteers who received two doses of subunit vaccine after live recombinant canarypox priming developed neutralizing antibodies directed against laboratory strains of HIV-1 and seven out of eight volunteers tested developed neutralizing antibodies to the primary isolate, BZ167, but to none of eight other primary isolates. Unprimed controls had low or absent neutralizing antibodies after two doses of subunit vaccine. CONCLUSIONS: The live canarypox vector was safe, stimulated cytotoxic T-cells and primed for a vigorous neutralizing antibody response upon boosting with subunit gp120 vaccine. This vaccine combination should be evaluated further for inducing protection against HIV infection.

Comparison of anti-HIV-1 ADCC reactivities in infected humans and chimpanzees.

Despite its shortcomings as a disease model, the chimpanzee is still the most relevant animal model for human immunodeficiency virus type 1 (HIV-1) infection. Previous studies have revealed qualitative differences between human and chimpanzee anti-HIV-1 responses. In this study, the development of specific anti-HIV-1 antibody-dependent cellular cytotoxic (ADCC) reactivities was evaluated in chronically infected chimpanzees and compared to the human response, because anti-HIV-1 ADCC represents a major component of anti-envelope cytolytic response found in infected patients. Ten HIV-1-infected chimpanzees up to 5 years after the infection were investigated. Anti-HIV-1 ADCC-directing antibodies were detectable in only three of 10 infected chimpanzees, and in these animals, activity was apparent only several months after the HIV infection. In some of the infected animals, ADCC reactivity against infected cells preceded reactivity against gp120-coated targets. When anti-gp120 ADCC-directing antibodies were apparent, they exhibited the same broad reactivity described in humans against different HIV isolates. The pattern of ADCC reactivities in infected chimpanzees is completely different from the well-characterized anti-gp120 cytotoxic reactivities present in HIV-1-infected patients. It is a relatively rare and late-occurring event that may have an important bearing on the lack of virus-induced pathogenesis in the chimpanzee model.