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Biomedical subjects

G P Mazzara

Publications and source records attributed to G P Mazzara.

At least 19 recordsLinked to original sources
PubMed2001

The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals.

The HIV-1 regulatory proteins Rev and Tat are expressed early in the virus life cycle and thus may be important targets for the immune control of HIV-1-infection and for effective vaccines. However, the extent to which these proteins are targeted in natural HIV-1 infection as well as precise epitopes targeted by human cytotoxic T lymphocytes (CTL) remain to be defined. In the present study, 57 HIV-1-infected individuals were screened for responses against Tat and Rev by using overlapping peptides spanning the entire Tat and Rev proteins. CD8+ T cell responses against Tat and Rev were found in up to 19 and 37% of HIV-1-infected individuals, respectively, indicating that these regulatory proteins are important targets for HIV-1-specific CTL. Despite the small size of these proteins, multiple CTL epitopes were identified in each. These data indicate that Tat and Rev are frequently targeted by CTL in natural HIV-1 infection and may be important targets for HIV vaccines.

CD8-Positive T-Lymphocytes↗
PubMed2000

Strategy for monitoring T cell responses to NY-ESO-1 in patients with any HLA class I allele.

NY-ESO-1 elicits frequent antibody responses in cancer patients, accompanied by strong CD8(+) T cell responses against HLA-A2-restricted epitopes. To broaden the range of cancer patients who can be assessed for immunity to NY-ESO-1, a general method was devised to detect T cell reactivity independent of prior characterization of epitopes. A recombinant adenoviral vector encoding the full cDNA sequence of NY-ESO-1 was used to transduce CD8-depleted peripheral blood lymphocytes as antigen-presenting cells. These modified antigen-presenting cells were then used to restimulate memory effector cells against NY-ESO-1 from the peripheral blood of cancer patients. Specific CD8(+) T cells thus sensitized were assayed on autologous B cell targets infected with a recombinant vaccinia virus encoding NY-ESO-1. Strong polyclonal responses were observed against NY-ESO-1 in antibody-positive patients, regardless of their HLA profile. Because the vectors do not cross-react immunologically, only responses to NY-ESO-1 were detected. The approach described here allows monitoring of CD8(+) T cell responses to NY-ESO-1 in the context of various HLA alleles and has led to the definition of NY-ESO-1 peptides presented by HLA-Cw3 and HLA-Cw6 molecules.

Alleles↗
PubMed2000

Identification of multiple simian immunodeficiency virus (SIV)-specific CTL epitopes in sooty mangabeys with natural and experimentally acquired SIV infection.

Host immune responses to SIV infection in sooty mangabeys are likely to be an important determinant of how such nonhuman primate species maintain asymptomatic lentivirus infection. We have previously described two patterns of asymptomatic SIV infection in sooty mangabeys: low viral loads with vigorous SIV-specific CTL activity in SIVmac239-infected sooty mangabeys, and high viral loads with generally weak or absent SIV-specific CTL activity in naturally infected sooty mangabeys. To define the specificity of the CTL response in SIV-infected mangabeys, we characterized CTL epitopes in two naturally infected and three SIVmac239-infected sooty mangabeys. Compared with that in SIVmac239-infected mangabeys, the yield of SIV-specific CTL clones was significantly lower in naturally infected sooty mangabeys. All CTL clones were phenotypically CD3+ CD8+, and lysis was MHC restricted. Seven SIV CTL epitopes were identified in five sooty mangabeys: one in Gag and three each in Nef and Envelope (Env). The CTL epitopes mapped to conserved regions in the SIV genome and were immunodominant. Several similar or identical CTL epitopes were recognized by both naturally infected and SIVmac239-infected mangabeys that shared class I MHC alleles. To our knowledge, this is the first report of SIV-specific CTL epitopes in sooty mangabeys. Longitudinal studies of viral load and sequence variation in CTL epitopes may provide useful information on the role of CTL in control or persistence of SIV infection in sooty mangabeys.

Amino Acid Sequence↗
PubMed1999

The use of combination vaccinia vaccines and dual-gene vaccinia vaccines to enhance antigen-specific T-cell immunity via T-cell costimulation.

Several recombinant vaccinia viruses are currently being evaluated to induce antigen-specific immunity to a variety of infectious disease agents and tumor associated antigens. T-cell costimulation is extremely important in enhancing T-cell responses, and recombinant vaccines have now been shown to be effective vectors to express a range of these molecules. Both combination vaccines (an admixture of a recombinant vaccinia virus expressing a specific target antigen and a recombinant vaccinia virus expressing a costimulatory molecule) and dual-gene vaccines expressing both transgenes on the same vector have been shown capable of effectively enhancing antigen-specific responses via T-cell costimulation. In this report, we compare for the first time the use of both types of approaches to enhance antigen-specific T-cell responses, and we demonstrate the importance of route of vaccine administration and vaccine dose in attaining optimal T-cell responses. These studies should have direct bearing on the design of vaccine clinical trials for infectious agents and/or tumor associated antigens, in which T-cell costimulatory molecules will be employed to enhance antigen-specific T-cell responses via the use of either combination or dual-gene vaccinia vaccines.

Adjuvants, Immunologic↗
PubMed1999

Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations.

Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Animals↗
PubMed1998

Cross-clade envelope glycoprotein 160-specific CD8+ cytotoxic T lymphocyte responses in early HIV type 1 clade B infection.

A major objective of current HIV-1 vaccination strategies is the induction of HIV-1-specific CD8+ MHC class I-restricted CTL responses, which are suggested to play a pivotal role in viral clearance and protection against HIV-1 disease progression. However, the marked genetic diversity of HIV-1 and existence of distinct viral subtypes or clades could potentially hinder the development of a universally efficacious HIV-1 vaccine. In this study we examined HIV-1 intraclade (B(LAI) versus B(MN)) Env gp160-specific CTL reactivity in recently HIV-1 clade B-infected individuals. We further evaluated the extent of interclade CTL cross-recognition of the divergent A and C Env gp160 subtypes, that are highly prevalent in the global pandemic. Freshly isolated PBMCs were stimulated in vitro with autologous PBMCs infected with recombinant vaccinia vectors expressing HIV-1 env, gag, pol, and nef genes derived from HIV-1 clade B. All 13 of the 19 HIV-1-seropositive subjects who elicited significant clade B Env gp160LAI CD8+ CTL responses also demonstrated comparable levels of CTL cross-reactivity against clade C92BR025 Env gp160. Nine of these individuals also showed extensive interclade CTL cross-recognition of clade A92UG037 Env gp160. Two HLA class I B7 donors had nondetectable intraclade CTL response against B Env gp160MN, while generating significant intraclade B(LAI) and interclade (A and C) Env gp160 CTL cross-reactivity. These observations serve to underscore the central importance of the HLA background of individuals in determining the pattern of immune reactivity to natural HIV-1 infection and presumably vaccines. Five donors studied also demonstrated broad CTL cross-reactivity against clade A92UG037 Gag p55, Pol, and/or Nef antigens. In conclusion, this present study indicates that there is a considerable degree of CD8+ CTL cross-recognition of the highly divergent HIV-1 Env gp160 subtypes during early phases of HIV-1 infection. Such findings suggest that HIV-1 vaccines based on a single clade that can induce extensive cross-clade immunity may demonstrate utility in diverse geographical regions.

CD8-Positive T-Lymphocytes↗
PubMed1997

Cytotoxic T-lymphocyte cross-reactivity among different human immunodeficiency virus type 1 clades: implications for vaccine development.

Despite recent advances in antiviral therapy for human immunodeficiency virus (HIV) infection, successful global intervention will require an effective vaccine. Expanding evidence suggests that cytotoxic T-lymphocyte (CTL) responses will be an important component of such a vaccine. The varying geographic distribution of HIV type 1 (HIV-1) clades, with the relative absence of clade B HIV-1 outside the developed world, is considered a major obstacle to the development of a single efficacious vaccine. An understanding of cross-reactive CTL responses between different HIV-1 clades is crucial in the design of a vaccine which will be broadly immunogenic. In this study, we examined the ability of HIV-1 Gag-, reverse transcriptase-, and Env-specific CTL clones isolated from individuals infected in the United States to recognize non-B clade viral sequences and found that all were cross-reactive with the majority of non-B clade viral sequences tested. We next studied HIV-1-specific CTL responses in African individuals infected with clade A, C, or G virus and evaluated cross-recognition of clade B virus. Of 14 persons evaluated, all demonstrated cross-reactivity with the U.S. clade B viral constructs. We conclude that significant CTL cross-reactivity exists between clade B and non-B epitopes, suggesting that CTL cross-recognition among HIV-1 clades is more widespread than anticipated and that a vaccine based on a single clade may be broadly applicable.

Amino Acid Sequence↗
PubMed1996

Use of a modified polysaccharide gel in developing a realistic breast phantom for MRI.

A polysaccharide material, TX-151, has been used together with water, NaCl, and Al powder to create a tissue equivalent gel to make a realistic, inexpensive, conveniently moldable, temporally stable tissue equivalent MRI phantom. Various phantom compositions were studied for variations in gelling time and relaxation times. Gd-DTPA added as a T1 (and T2) modifier and aluminum powder added to decrease T2 permitted phantoms to be made with a range of relaxation times comparable to human tissues. We have used this polysaccharide gel to create breast phantoms for testing breast coils and evaluating different MRI imaging sequences available for diagnosis. The breast phantoms consisted of a layer of Crisco, a good model for adipose tissue, surrounding the TX-151 gel. Some of these phantoms were created with a silicone implant encapsulated in the gel to simulate an augmented breast. More sophisticated phantoms can easily be developed by additions of other materials to this polysaccharide gel.

Female↗
PubMed1994

High-titer immune responses elicited by recombinant vaccinia virus priming and particle boosting are ineffective in preventing virulent SIV infection.

Eighteen rhesus monkeys were vaccinated with recombinant vaccinia viruses expressing SIVmac antigens in 3 separate rounds of experiments. Twelve of the monkeys were primed with a trivalent vaccinia virus recombinant expressing Gag, Pol, and Env polypeptides that can assemble into SIV pseudovirion particles and boosted with SIV particles in adjuvant. Four of the monkeys were primed with different vaccinia virus recombinants expressing env or gag+env followed by SIV particle boosts; two received vaccinia virus recombinants alone (env or env+gag). Despite the induction of vigorous immune responses, 17 of 18 rhesus monkeys became infected on challenge with a low dose of virulent SIVmac. The single protected animal was one of three challenged with homologous cloned SIV exactly matched to the clone used for construction of trivalent vaccinia virus recombinant and particles. Vaccination may have diminished SIV burdens and rates of CD4+ cell declines in some of the animals, but vaccinated/challenge/infected animals eventually developed fatal disease similar to control animals. These results highlight the extreme difficulty in achieving vaccine protection against virulent SIVmac infection even under idealized laboratory conditions.

Animals↗
PubMed1993

Immunization with lentivirus-like particles elicits a potent SIV-specific recall cytotoxic T-lymphocyte response in rhesus monkeys.

An effective acquired immunodeficiency syndrome (AIDS) vaccine should be capable of eliciting human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) responses. We have explored the use of lentivirus-like particles produced in mammalian cells infected with a recombinant vaccinia virus to immunize for the induction of CTL in the simian immunodeficiency virus (SIV)/rhesus monkey model for AIDS. SIV-like particles in a threonyl-MDP-based adjuvant did not elicit a high frequency of SIV gag-specific effector cells in naive rhesus monkeys. However, immunization with these particles elicited a potent recall CTL response in monkeys previously vaccinated with a recombinant vaccinia-SIV virus. These observations suggest that poxvirus-produced virus-like particles may represent a safe immunogen for use in periodic boosting to maintain viable cell-mediated immunity to the AIDS virus.

Animals↗
PubMed1991

Limiting dilution analysis of cytotoxic T lymphocytes to human immunodeficiency virus gag antigens in infected persons: in vitro quantitation of effector cell populations with p17 and p24 specificities.

The presence of cytotoxic T lymphocytes (CTL) to the gag antigens of human immunodeficiency virus (HIV) has been described in infected populations. We found that the majority of this immune response as measured in bulk CTL assays of unstimulated peripheral blood mononuclear cells (PBMC) is directed against the p24 component of the p55 gag precursor protein. Using limiting dilution analysis of this effector cell population we confirm that the majority of activated gag-specific CTL circulating in the PBMC of infected hemophilic patients are directed at p24 determinants and are present at frequencies of 1/36,000 to 1/86,000 lymphocytes. By performing in vitro stimulation after limiting dilution, the precursor population of gag-specific CTL are characterized and quantitated. HIV gag-specific CTL precursors are identified at frequencies of 1/1700 to 1/17,000 lymphocytes and are made up of cells with both p17 and p24 specificities. No HIV gag-specific CTL precursor cells are identified in the PBMC of HIV-uninfected individuals. These studies demonstrate that CTL directed at both p17 and p24 determinants make up the cellular immune repertoire in HIV-infected individuals but that only the p24-specific CTL are routinely found in an activated state in the circulation.

CD8 Antigens↗
PubMed1991

HIV-1 gag-specific cytotoxic T lymphocytes recognize multiple highly conserved epitopes. Fine specificity of the gag-specific response defined by using unstimulated peripheral blood mononuclear cells and cloned effector cells.

CTL directed at the highly conserved HIV-1 gag protein have been described in HIV-1 seropositive persons and may be an important host defense against this retrovirus. Presently only limited data are available regarding the specific epitopes recognized by these CTL. In this study, we have performed a detailed examination of the gag-specific CTL response in three HIV-1 seropositive subjects, using both unstimulated PBMC and cloned CTL. Lysis of gag-expressing targets was found to be mediated by CD3+CD8+ lymphocytes and restricted by class I Ag. Multiple class I Ag were found to restrict gag epitopes in each subject studied, with as many as three of these Ag involved in presenting gag CTL epitopes in a single subject. The majority of gag-specific CTL activity was found to be directed against epitopes in the p24 subunit of the gag protein, with at least seven different HLA class I-restricted CTL p24 epitopes identified in these three subjects. Less CTL activity was directed against p17 subunit of gag and two CTL epitopes were identified in this protein. Although as many as four different epitopes in gag were recognized using CTL from a single subject, none of the epitopes was recognized by CTL from more than one subject. Analysis of gag epitope recognition using cloned CTL demonstrated heterogeneity and specificity not appreciated using unstimulated PBMC. The identification of multiple relatively conserved epitopes in the HIV-1 gag protein and the heterogeneity of CTL responses to this protein may have important implications for vaccine development and our understanding of AIDS pathogenesis.

Amino Acid Sequence↗
PubMed1991

Recombinant virus vaccine-induced SIV-specific CD8+ cytotoxic T lymphocytes.

Evidence indicates that cytotoxic T lymphocytes (CTLs) may be important in containing the spread of the human immunodeficiency virus (HIV) in the infected host. Although the use of recombinant viruses has been proposed as an approach to elicit protective immunity against HIV, the ability of recombinant viral constructs to elicit CD8+ CTL responses in higher primates has never been demonstrated. A live recombinant virus, vaccinia-simian immunodeficiency virus of macaques (SIVmac), was used to determine whether such a genetically restricted, T lymphocyte-mediated antiviral response could be generated in a primate. Vaccinia-SIVmac vaccination elicited an SIVmac Gag-specific, CD8+ CTL response in rhesus monkeys. These CTLs recognized a peptide fragment that spans residues 171 to 195 of the Gag protein. The rhesus monkey major histocompatibility complex (MHC) class I gene product restricting this CTL response was defined. Both the vaccinated and SIVmac-infected monkeys that shared this MHC class I gene product developed CTLs with the same Gag epitope specificity. These findings support the use of recombinant virus vaccines for the prevention of HIV infections in humans.

Amino Acid Sequence↗
PubMed1990

Two distinct lymphocyte populations mediate simian immunodeficiency virus envelope-specific target cell lysis.

The relative contributions of the effector lymphocyte responses to the AIDS virus envelope glycoprotein (env) were explored in simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys. CD8+, MHC class I-restricted, env-specific CTL were cloned from PBL of SIVmac-infected monkeys, indicating that such cells constitute a component of the env-specific effector lymphocyte response. A limiting dilution 51Cr release assay was then established for quantitating the frequency of SIVmac-specific effector lymphocytes in PBL of rhesus monkeys. Using this assay we demonstrate that SIVmac env-specific effector lymphocytes are comprised of both CD16-, MHC class I-restricted and CD16+, MHC class I-unrestricted cells. We also demonstrate that the env-specific response is the predominant SIVmac-specific effector lymphocyte response in rhesus monkeys. These studies document the complexity of the effector lymphocyte response to the AIDS virus envelope glycoprotein and establish the role played by two distinct effector cell populations in this response.

Animals↗
PubMed1990

Studies of cloned simian immunodeficiency virus-specific T lymphocytes. gag-specific cytotoxic T lymphocytes exhibit a restricted epitope specificity.

CD8+ CTL inhibit the replication of HIV and simian immunodeficiency virus of macaques (SIVmac) in PBL and, therefore, are likely to play an important role in containing the spread of the AIDS virus in infected individuals. We have generated a series of gag-specific lytic T lymphocyte clones from PBL: of an SIVmac-infected rhesus monkey. These T cell clones are CD3+CD8+ and are MHC class I-restricted in their target specificity. They are, therefore, CTL. Interestingly, all gag-specific CTL clones, as well as the gag-specific lytic activity of PBL of this monkey, demonstrated specificity for a single 25 amino acid fragment of the SIVmac gag protein. Moreover, they were restricted in their lytic function by a single MHC class I allele. These findings illustrate a powerful method for cloning AIDS virus-specific T lymphocytes and demonstrate a remarkably restricted epitope specificity of this AIDS virus-specific CTL response.

Animals↗
PubMed1990

The gag-specific cytotoxic T lymphocytes in rhesus monkeys infected with the simian immunodeficiency virus of macaques.

The simian immunodeficiency virus of macaques (SIVmac) is a lentivirus which induces an AIDS-like disease in rhesus monkeys. We have explored the virus-specific cellular immune response in SIVmac-infected rhesus monkeys. Con A-activated, IL-2 expanded PBL of some SIVmac-infected rhesus monkeys lyse autologous B lymphoblastoid cell lines infected with a recombinant vaccinia virus that carries the SIVmac gag gene. This lysis is mediated by CD8+ lymphocytes and is MHC class I restricted. Moreover, these effector lymphocytes do not express the NK cell-associated molecules NKH1 or CD16. These cells are, therefore, CTL. In a limited prospective study of SIVmac-infected rhesus monkeys, the presence of the SIVmac gag-specific CTL activity in PBL correlated with both a reduced efficiency in isolating SIVmac from PBL of these monkeys and their extended survival. This method for assessing SIVmac gag-specific cellular immunity in rhesus monkeys will be important not only in investigating the immunopathogenesis of SIVmac-induced disease, but also in evaluating the capacity of candidate AIDS vaccines to elicit a cell-mediated immune response in this animal model.

Animals↗