Mark J. Mulligan

A significant number of human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma interferon.

ABSTRACT Despite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8+ T lymphocytes are discussed.

A Canarypox Vaccine Expressing Multiple Human Immunodeficiency Virus Type 1 Genes Given Alone or with Rgp120 Elicits Broad and Durable CD8+ Cytotoxic T Lymphocyte Responses in Seronegative Volunteers

Induction of CD8+ cytotoxic T cells is considered one of the important correlates for the protective efficacy of candidate human immunodeficiency virus type 1 (HIV-1) vaccines. To induce CD8+ cytotoxic T lymphocytes (CTLs) along with neutralizing antibody and CD4+ T cell help, a live canarypox virus construct expressing gp120, transmembrane gp41, the gag and protease genes, and sequences containing CTL epitopes in nef and pol was given simultaneously with, or followed by, rgp120 SF2. CD8+ CTLs were detected in 61% of volunteers at some time during the trial. Three to 6 months after the last immunization, the gene-specific responses were gag, 26/81; env, 17/77; nef, 12/77; and pol, 3/16. Simultaneous immunization with the canarypox vector and the subunit, beginning with the initial immunization, resulted in earlier antibody responses. In summary, a strategy of immunization with a canarypox vector expressing multiple genes of HIV-1 given with gp120 results in durable CD8+ CTL responses to a broad range of epitopes.

Safety and Immunogenicity of a Canarypox-Vectored Human Immunodeficiency Virus Type 1 Vaccine with or without gp120: A Phase 2 Study in Higher- and Lower-Risk Volunteers

Live attenuated viral vectors that express human immunodeficiency virus (HIV) antigens are being developed as potential vaccines to prevent HIV infection. The first phase 2 trial with a canarypox vector (vCP205, which expresses gp120, p55, and protease) was conducted in 435 volunteers with and without gp120 boosting, to expand the safety database and to compare the immunogenicity of the vector in volunteers who were at higher risk with that in volunteers at lower risk for HIV infection. Neutralizing antibodies to the MN strain were stimulated in 94% of volunteers given vCP205 plus gp120 and in 56% of volunteers given vCP205 alone. CD8(+) cytotoxic T lymphocyte cells developed at some time point in 33% of volunteers given vCP205, with or without gp120. Phase 3 field trials with these or similar vaccines are needed, to determine whether efficacy in preventing HIV infection or in slowing disease progression among vaccinees who become infected is associated with the level and types of immune responses that were induced by the vaccines in this study.

QS-21 promotes an adjuvant effect allowing for reduced antigen dose during HIV-1 envelope subunit immmunization in humans

Three separate studies were undertaken in HIV-1 uninfected persons to determine if the adjuvant QS-21 improves the magnitude or kinetics of immune responses induced by recombinant soluble gp120 HIV-1(MN) protein (rsgp120) immunization. The QS-21 was administered at two doses (50 and 100 microg), either alone or in combination with aluminum hydroxide (600 microg). At the highest doses of rsgp120 (100, 300, and 600 microg), QS-21 exerted no significant effect on either binding or neutralizing antibody titers. Antibody binding and neutralizing responses fell dramatically when rsgp120, formulated with alum alone, was given at low doses (3 and 30 microg). In contrast, antibody responses similar in titer to those in the high dose antigen groups were induced with the low dose rsgp120 formulated with QS-21. In addition, the lymphocyte proliferation and delayed type hypersensitivity skin testing were superior in the QS-21 recipients compared with the alum recipients at the low antigen doses. Moderate to severe pain was observed in majority of the volunteers receiving QS-21 formulations, and vasovagal episodes and hypertension were not infrequent. Thus, the use of QS-21 may provide a means to reduce the dose of a soluble protein immunogen.

Phase 2 Study of an HIV-1 Canarypox Vaccine (vCP1452) Alone and in Combination With rgp120: Negative Results Fail to Trigger a Phase 3 Correlates Trial

Background:A goal of T-cell HIV vaccines is to define the correlation between a vaccine-induced immune response and protection from HIV infection. We conducted a phase 2 trial to determine if a canarypox vaccine candidate (vCP1452) administered with rgp120 subunit protein would “qualify” for a trial to define a correlate of efficacy. Methods:A total of 330 healthy volunteers were enrolled into 4 groups: 120 received vCP1452 alone (0, 1, 3, and 6 months), 120 received vCP1452 with 2 different regimens of rgp120 coadministration, and 90 received placebo. HIV-specific antibody responses were measured by enzyme-linked immunoassay (ELISA) and neutralizing activity. T-cell responses were measured by chromium release and interferon-γ (IFNγ) enzyme-linked immunospot (ELISpot) assay. Results:Significant neutralizing antibody responses to the HIV MN strain were detected in all vaccine groups, with net responses ranging from 57% (95% confidence interval [CI]: 40% to 71%) to 94% (95% CI: 85% to 99%). Net cumulative HIV-specific CD8+ IFNγ ELISpot assay responses were 13% (95% CI: −1% to 26%) for recipients of vCP1452 alone and 16% (95% CI: 2% to 29%) for recipients of vCP1452 plus rgp120. Conclusions:Overall, the HIV-specific CD8+ cytotoxic T lymphocyte (CTL) response was not sufficient to qualify the regimen for a subsequent trial designed to detect an immune correlate of protection requiring a minimum CD8+ CTL frequency of 30%.

Polymorphisms in HLA Class I Genes Associated with both Favorable Prognosis of Human Immunodeficiency Virus (HIV) Type 1 Infection and Positive Cytotoxic T-Lymphocyte Responses to ALVAC-HIV Recombinant Canarypox Vaccines

ABSTRACT Carriers of certain human leukocyte antigen class I alleles show favorable prognosis of human immunodeficiency virus type 1 (HIV-1) infection, presumably due to effective CD8+ cytotoxic T-lymphocyte responses, but close relationships between class I variants mediating such responses to natural and to vaccine HIV-1 antigen have not been established. During 6 to 30 months of administration and follow-up in trials of ALVAC-HIV recombinant canarypox vaccines, cells from 42% of 291 HIV-1-negative vaccinated subjects typed at class I loci responded to an HIV-1 protein in a lytic bulk CD8+ cytotoxic T-lymphocyte assay. By 2 weeks after the second dose, higher proportions of vaccinees carrying one of two alleles consistently associated with slower progression of natural HIV-1 infection reacted at least once: B∗27 carriers reacted to Gag (64%; odds ratio [OR] = 10.3, P = 0.001) and Env (36%; OR = 4.6, P = 0.04), and B∗57 carriers reacted to Env (44%; OR = 6.6, P < 0.05). By 2 weeks after the third or fourth dose, B∗27 carriers had responded (two or more reactions) to Gag (33%; OR = 4.4, P< 0.05) and B∗57 carriers had responded to both Gag (39%; OR = 5.3, P = 0.013) and Env (39%; OR = 9.5,P = 0.002). Homozygosity at class I loci, although conferring an unfavorable prognosis following natural infection, showed no such disadvantage for vaccine response. Individual class I alleles have not previously demonstrated such clear and consistent relationship with both the clinical course of an infection and cellular immunity to a vaccine against the infectious agent. This proof of principle that class I an alleles modulate both processes has implications for development of HIV-1 and presumably other vaccines.

Host genetic profiles predict virological and immunological control of HIV-1 infection in adolescents

Objective: To evaluate the correlation between host genetic profiles and virological and immunological outcomes among HIV-1-seropositive participants from the Reaching for Excellence in Adolescent Care and Health (REACH) cohort. Methods: HLA class I and chemokine coreceptor (CCR) alleles and haplotypes were resolved in 227 HIV-1-seropositive adolescents (ages 13–18 years; 75% females; 71% African-Americans) and 183 HIV-seronegative individuals, with quarterly follow-up visits between 1996 and 2000. Each HLA and CCR variant with consistent risk and protective effect on HIV-1 pathogenesis was assigned a score of −1 and +1, respectively. All individual markers and genetic scores were analyzed in relation to plasma viral load (VL) and CD4 T lymphocytes during a 6–12-month interval when no antiretroviral therapy was taken. Results: HLA-B*57 alone was a strong predictor of VL (P < 0.0001), but composite genetic profiles found in over 50% of patients consistently outperformed the individual component markers in multivariable analyses with or without adjustment for gender, race, age, and membership of clinical patient groups. Adolescents (n = 37) with a favorable combination of VL (< 1000 copies/ml) and CD4 T cell counts (> 450 × 106 cells/l) consistently had more positive (+1 to +2) than negative (−1 to −4) HLA and CCR scores compared with those (n = 56) with an unfavorable combination (VL > 16 000 copies/ml and CD4 cells < 450 × 106 cells/l) or the remainder (n = 134) of the cohort (overall P < 0.0001). Conclusion: A generalizable genetic scoring algorithm based on seven HLA class I and CCR markers is highly predictive of viremia and immunodeficiency in HIV-1-infected adolescents.

Identification of an ER retrieval signal in a retroviral glycoprotein

Soluble or membrane-spanning proteins that are resident in the endoplasmic reticulum (ER) possess short amino acid sequence motifs that result in their localization in the ER by retrieval, retention, or both. One such protein targeting signal, the carboxy-terminal tetrapeptide KDEL (Munro and Pelham, 1987), retrieves soluble proteins from the Golgi complex to the ER lumen by interacting with the KDEL receptor (Lewis and Pelham, 1992). A second consensus motif, consisting of two lysines located at positions -3 and either-4 or-5 from the cytoplasmic carboxyl terminus, was identified in 14 of 15 ER resident type 1 membrane proteins (Jackson et al., 1990) (Table 1). Proteins with the dilysine signal are retrieved from postER compartments to the ER (Jackson et al., 1993) by cytosolic coat proteins (COPS) controlling retrograde vesicular transport (Cosson and Letourneur, 1994; Letourneur et al., 1994). Type 2 membrane proteins that reside in the ER possess an analogous double-arginine ER localization signal at their cytoplasmic amino termini (Schutze et al., 1994). Enveloped viruses bud through the cellular membrane to which their glycoproteins sort (reviewed by Stephens and Compans, 1988). Some viral glycoproteins sort to the specialized apical or basolateral plasma membrane domains of polarized epithelial cells and direct virion budding across those membranes. For example, gp160 of human immunodeficiencyvirus type 1 (HIV-l) sorts to the basolat-eral domain (Owens et al., 1991; Lodge et al., 1994), while hemagglutinin of influenzavirus sorts to the apical domain (Roth et al., 1983). To mediate budding of infectious viral particles through intracellular membranes, viral glycopro-teins must fulfill two requirements: a signal for sorting to an intracellular compartment and an interaction with the core proteins of the virus. For example, membrane-spanning domains of the M protein of coronaviruses (Ma-chamer and Rose, 1987; Swift and Machamer, 1991) or the Gl protein of bunyaviruses (Matsuoka et al., 1994) specify glycoprotein accumulation at the Golgi complex. The adenovirus glycoprotein El9 possesses adilysine signal that localizes it to the ER (Jackson et al., 1990) where it binds class I molecules to diminish recognition of adeno-Letter to the Editor virus-infected cells by cytotoxic T lymphocytes (Cox et al., 1991). The foamy viruses are a genus of retroviruses that infect a wide variety of mammalian hosts; e.g., they are ubiquitous in nonhuman primates, bovines, and felines and occasionally infect humans and other mammals (Hooks and Gibbs, 1975). However, foamy viruses have yet to be associated definitively with any disease process Similar …

A Phase I safety and immunogenicity trial of UBI® microparticulate monovalent HIV-1 MN oral peptide immunogen with parenteral boost in HIV-1 seronegative human subjects

Thirty-three HIV-seronegative adults were recruited into a Phase I safety and immunogenicity HIV-1 vaccine trial. The immunogens were as follows: a synthetic, monovalent, octameric HIV-1 MN V3 peptide in aluminum hydroxide (alum) adjuvant administered by intramuscular delivery; and a similar product encapsulated in biodegradable micro-spheres composed of co-polymers of lactic and glycolic acids, administered by the oral route. These were administered in three sequential oral doses, followed by a parenteral boost. No serious adverse experiences were observed. Oral administration of this vaccine, alone or in combination with parenteral boosting, resulted in no significant humoral, cellular, or mucosal immune responses.

A POLARIZED HUMAN ENDOMETRIAL CELL LINE THAT BINDS AND TRANSPORTS POLYMERIC IgA

SummaryWe have demonstrated that a human endometrial cell line, HEC-1, maintains a high transepithelial electrical resistance, directionally transports fluids across the cell monolayer, and releases enveloped viruses at distinct plasma membrane domains: influenza virus is released at the apical surfaces and vesicular stomatitis virus (VSV) at the basolateral surfaces. In addition, we have examined the expression of domain-specific endogenous proteins, including the polyimmunoglobulin receptor. Multiple endogenous polypeptides were found to be secreted into the culture medium at basolateral surfaces, whereas no secretion of specific polypeptides was observed from apical cell surfaces. Distinct patterns of endogenous proteins were also observed on apical and basolateral cell surfaces, with a much more complex polypeptide pattern on the basolateral membranes. Using surface biotinylation and immunofluorescence, the polyimmunoglobulin receptor was found to be expressed on the basolateral surfaces of HEC-1 monolayers. The specific binding of poly-immunoglobulin A (pIgA) was found to occur on the basolateral surface, and was followed by transcytosis to the apical surface and release into the apical medium. The observed characteristics indicate that the endometrium-derived HEC-1 epithelial cell line can be employed as a model for studies of protein transport in polarized epithelial cells of human endometrial tissues, as well as for studies of the interaction of microorganisms with epithelial cells in the genital tract.