Surface antigens of Toxoplasma gondii: variations on a theme.

Toxoplasma gondii is an obligate intracellular protozoan parasite with an exceptionally broad host range. Recently, it has become apparent that the number of surface antigens (SAGs) it expresses may rival the number of genera it can infect. Most of these antigens belong to the developmentally regulated and distantly related SAG1 or SAG2 families. The genes encoding the surface antigens are distributed throughout the T. gondii genome, with remarkably little polymorphism observed at each locus. Results from a number of studies have suggested that the surface antigens play an important role in the biology of the parasite. For example, SAG3 null mutants generated by targeted disruption provide convincing evidence that this surface antigen, at least, functions during parasite attachment. Analyses of a SAG1 knockout in rodents, however, indicate that this surface antigen may play a crucial role in immune modulation or virulence attenuation. The current understanding of the SAG1 and SAG2 families will be discussed here.

Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells.

Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.

Toxoplasma gondii: identification of a developmentally regulated family of genes related to SAG2.

Previous studies have shown the surface of Toxoplasma gondii to be dominated by a family of proteins closely related to SAG1. In this study, we report the existence of a second family of genes defined by homology to SAG2. The predicted amino acid sequences of these three new proteins suggests that they are all glycosylphosphatidylinositol-linked surface antigens. All three also contain N-linked glycosylation sites, although their use has yet to be demonstrated. One of these SAG2-related antigens, SAG2B, is expressed in tachyzoites with an apparent size of 23 kDa. It is distinct, however, from the previously identified P23. In contrast to SAG2B, SAG2C and SAG2D appear to be expressed exclusively on the surface of bradyzoites. Analysis of the SAG2 family shows it to have weak but significant homology to the SAG1 family. Thus, all of the sequenced surface antigens of tachyzoites and many of those of bradyzoites fall into one large superfamily that can be divided into two subgroups defined by the prototypic and highly immunogenic SAG1 and SAG2, respectively.

Human immunodeficiency virus type 1 envelope epitope-specific CD4(+) T lymphocytes in simian/human immunodeficiency virus-infected and vaccinated rhesus monkeys detected using a peptide-major histocompatibility complex class II tetramer.

A tetrameric recombinant major histocompatibility complex (MHC) class II-peptide complex was used to quantitate human immunodeficiency virus type 1 (HIV-1) envelope (Env)-specific CD4(+) T cells in vaccinated and in simian/human immunodeficiency virus (SHIV)-infected rhesus monkeys. A rhesus monkey MHC class II DR molecule, Mamu-DR*W201, and an HIV-1 Env peptide (p46) were employed to construct this tetrameric complex. A p46-specific proliferative response was seen in sorted, tetramer-binding, but not nonbinding, CD4(+) T cells, directly demonstrating that this response was mediated by the epitope-specific lymphocytes. Although staining of whole blood from 10 SHIV-infected Mamu-DR*W201(+) rhesus monkeys failed to demonstrate tetramer-binding CD4(+) T cells (<0.02%), p46-stimulated peripheral blood mononuclear cells (PBMCs) from 9 of these 10 monkeys had detectable p46 tetramer-binding cells, comprising 0.5 to 15.2% of the CD4(+) T cells. p46-stimulated PBMCs from 7 of 10 Mamu-DR*W201(+) monkeys vaccinated with a recombinant canarypox virus-HIV-1 env construct also demonstrated p46 tetramer-binding cells, comprising 0.9 to 7.2% of the CD4(+) T cells. Thus, Env p46-specific CD4(+) T cells can be detected by tetrameric Mamu-DR*W201-p46 complex staining of PBMCs in both SHIV-infected and vaccinated rhesus monkeys. These epitope-specific cell populations appear to be present in peripheral blood at a very low frequency.

Substitutions in a major histocompatibility complex class II-restricted human immunodeficiency virus type 1 gp120 epitope can affect CD4+ T-helper-cell function.

It has been suggested that the inability of the immune response to control human immunodeficiency virus type 1 (HIV-1) replication may be due, at least in part, to the capacity of this virus to escape from immune recognition through mutation. While there is increasing evidence for the importance of HIV-1-specific CD4+ T cells in containing HIV-1 spread in the infected individual, little is known about the consequences of HIV-1 mutation on virus-specific CD() T-cell function. The impact of HIV-1 sequence variation on CD4+ T-helper (Th)- cell function was assessed with a rhesus monkey model for immune recognition of the HIV-1 envelope (Env) glycoprotein. A series of HIV-1 Env(484-496) variant peptides were shown to retain the ability to bind to the appropriate rhesus monkey major histocompatibility complex class II DR molecule. Peptides bearing substitutions at position 490, however, failed to drive the proliferation or cytokine secretion of two well-characterized HXBc2 Env-specific rhesus monkey CD4+ Th-cell lines. Exogenous costimulation was ineffective in complementing the ability of the nonstimulatory peptides to induce [3H]thymidine incorporation by these cells. Finally, HIV-1 Env(484-496) variant peptides with substitutions at position 490 antagonized the HXBc2 Env peptide-induced proliferative response of the CD4+ Th-cell lines. Thus, HIV-1 variants appear to have the capacity to neutralize the function of virus-specific CD4+ T lymphocytes.

HIV-1 envelope-specific CD4+ T helper cells from simian/human immunodeficiency virus-infected rhesus monkeys recognize epitopes restricted by MHC class II DRB1*0406 and DRB*W201 molecules.

The HIV-1 envelope (Env)-specific Th cell response of rhesus monkeys infected with nonpathogenic chimeric simian/human immunodeficiency viruses (SHIV) was assessed at a molecular level by establishing rgp120-specific CD4+ T cell lines. Epitopes recognized by these MHC class II-restricted Th cells were identified within the second (C2), third (C3), and fifth (C5) conserved regions of HIV-1 gp120. The epitope located in C2 was found to be restricted by the rhesus monkey (Macaca mulatta) leukocyte Ag Mamu-DRB1*0406, and the Th cell epitope in C5 was found to be restricted by Mamu-DRB*W201. The restriction element requirements of the C2 and C5 epitope-specific CD4+ Th cells appear to be rather stringent, because these peptide epitopes were not recognized in the presence of other Mamu-DR expressing cell lines. The ability to analyze HIV-1 Env-specific CD4+ T cell responses in SHIV-infected monkeys will enhance the utility of this model for studying AIDS pathogenesis and for assessing novel HIV-1 vaccine strategies.

Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination.

It is generally thought that an effective vaccine to prevent HIV-1 infection should elicit both strong neutralizing antibody and cytotoxic T lymphocyte responses. We recently demonstrated that potent, boostable, long-lived HIV-1 envelope (Env)-specific cytotoxic T lymphocyte responses can be elicited in rhesus monkeys using plasmid-encoded HIV-1 env DNA as the immunogen. In the present study, we show that the addition of HIV-1 Env protein to this regimen as a boosting immunogen generates a high titer neutralizing antibody response in this nonhuman primate species. Moreover, we demonstrate in a pilot study that immunization with HIV-1 env DNA (multiple doses) followed by a final immunization with HIV-1 env DNA plus HIV-1 Env protein (env gene from HXBc2 clone of HIV IIIB; Env protein from parental HIV IIIB) completely protects monkeys from infection after i.v. challenge with a chimeric virus expressing HIV-1 env (HXBc2) on a simian immmunodeficiency virusmac backbone (SHIV-HXBc2). The potent immunity and protection seen in these pilot experiments suggest that a DNA prime/DNA plus protein boost regimen warrants active investigation as a vaccine strategy to prevent HIV-1 infection.

HIV-1 env DNA vaccine administered to rhesus monkeys elicits MHC class II-restricted CD4+ T helper cells that secrete IFN-gamma and TNF-alpha.

The Th cell response elicited by an HIV-1 env plasmid DNA vaccine was assessed in rhesus monkeys by isolation of gp120-specific, MHC class II-restricted CD4+ T cell lines from PBL of vaccinated animals. The Env-specific CD4+ T cell lines recognized epitopes located in the second hypervariable region and in the carboxyl terminus of HIV-1 gp120. These cell lines proliferated in response to APC in the presence of recombinant gp120, as well as to APC expressing virally encoded Env. All of the CD4+ T cell lines responded to Env peptide by secreting IFN-gamma and TNF-alpha without appreciable IL-4 production. Recombinant gp120 stimulation of PBL from these vaccinated monkeys elicited the secretion of a similar profile of cytokines. Demonstration of a nucleotide vaccine eliciting a Th1-like immune response is consistent with the well documented ability of naked DNA vaccines to induce durable CD8+ CTL responses.

An env gene derived from a primary human immunodeficiency virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys.

To explore the roles played by specific human immunodeficiency virus type 1 (HIV-1) genes in determining the in vivo replicative capacity of AIDS viruses, we have examined the replication kinetics and virus-specific immune responses in rhesus monkeys following infection with two chimeric simian/human immunodeficiency viruses (SHIVs). These viruses were composed of simian immunodeficiency virus SIVmac239 expressing HIV-1 env and the associated auxiliary HIV-1 genes tat, vpu, and rep. Virus replication was assessed during primary infection of rhesus monkeys by measuring plasma SIVmac p27 levels and by quantifying virus replication in lymph nodes using in situ hybridization. SHIV-HXBc2, which expresses the HIV-1 env of a T-cell-tropic, laboratory-adapted strain of HIV-1 (HXBc2), replicated well in rhesus monkey peripheral blood leukocytes (PBL) in vitro but replicated only to low levels when inoculated in rhesus monkeys. In contrast, SHIV-89.6 was constructed with the HIV-1 env gene of a T-cell- and macrophage-tropic clone of a patient isolate of HIV-1 (89.6). This virus replicated to a lower level in monkey PBL in vitro but replicated to a higher degree in monkeys during primary infection. Moreover, monkeys infected with SHIV-89.6 developed an inversion in the PBL CD4/CD8 ratio coincident with the clearance of primary viremia. The differences in the in vivo consequences of infection by these two SHIVs could not be explained by differences in the immune responses elicited by these viruses, since infected animals had comparable type-specific neutralizing antibody titers, proliferative responses to recombinant HIV-1 gp120, and virus-specific cytolytic effector T-cell responses. With the demonstration that a chimeric SHIV can replicate to high levels during primary infection in rhesus monkeys, this model can now be used to define genetic determinants of HIV-1 pathogenicity.

Simian immunodeficiency virus-specific cytotoxic T-lymphocyte induction through DNA vaccination of rhesus monkeys.

In view of the growing evidence that virus-specific cytotoxic T lymphocytes (CTL) play an important role in containing the early spread of human immunodeficiency virus type 1 (HIV-1) in infected individuals, novel vaccine strategies capable of eliciting HIV-1-specific CTL are being pursued in attempts to create an effective AIDS vaccine. We have used the simian immunodeficiency virus of macaques (SIVmac)/rhesus monkey model to explore the induction of AIDS virus-specific CTL responses by DNA vaccination. We found that the inoculation of rhesus monkeys with plasmid DNA encoding SIVmac Env and Gag elicited a persisting SIVmac-specific memory CTL response. These CTL were CD8+ and major histocompatibility complex class I restricted. These studies provide evidence for the potential utility of DNA inoculation as an approach to an HIV-1 vaccine.

T cell receptor V beta repertoire in an acute infection of rhesus monkeys with simian immunodeficiency viruses and a chimeric simian-human immunodeficiency virus.

Changes in T cell receptor (TCR) V beta repertoire and their correlation with virologic events were investigated in rhesus monkeys after acute infection with the simian immunodeficiency virus (SIV). 11 genetically defined rhesus monkeys were experimentally infected with SIVmac or a chimeric simian-human immunodeficiency virus (SHIV), and their peripheral blood lymphocytes (PBL) and lymph nodes were prospectively assessed for TCR V beta gene expression. PBL and lymph nodes of the acutely infected monkeys demonstrated an expansion of selected V beta-expressing T lymphocyte subpopulations as early as 3 d after infection. These expanded V beta-expressing lymphocyte subpopulations were comprised predominantly of CD8+ cells. Six of seven infected monkeys sharing a single electrophoretically defined major histocompatibility complex class I allele exhibited a similar expansion of V beta 14-expressing PBL. Sequence analyses of V-D-J segments of TCR-beta cDNA indicated that the V beta-expressing T cell subpopulation expansion can be oligoclonal. SIVmac-specific CD8+ cytotoxic T lymphocytes were demonstrated in both PBL and lymph nodes of the infected monkeys at the time expansion of the selected V beta-expressing cell subpopulations was seen. Finally, the expansion of the selected V beta-expressing lymphocytes in PBL coincided with the emergence and clearance of SIV p27 from the plasma of the infected monkeys. These results demonstrate that acute infection of rhesus monkeys with SIVmac or SHIV results in an expansion of CD8+ lymphocyte subpopulations expressing selected V beta gene families. The selectively expanded T lymphocytes may contribute to early viral clearance after acute SIVmac or SHIV infection.

Biochemical and molecular characterization of rhesus monkey major histocompatibility complex class II DR.

Rapid, simple techniques for MHC class II typing were explored to facilitate the use of a wide variety of nonhuman primate species as models of human diseases and therapies. We demonstrate that radioimmunoprecipitation and 1-D IEF or 1-D NEPHGE can be employed for characterizing MHC class II DR alleles in rhesus monkeys. Complementary molecular analyses have yielded the first full-length nonhuman primate DRA sequence and the first full-length rhesus macaque DRB sequences. In this way it has also been possible to determine which subset of the DRB sequences amplified from a B-LCL are actually expressed.

MHC expression of African green monkeys of Barbados is limited in heterogeneity.

Major histocompatibility complex expression of activated peripheral blood lymphocytes of captive African green monkeys from Barbados and from Africa were analyzed biochemically; class I molecules by one-dimensional isoelectric focusing and class II DR molecules by one-dimensional nonequilibrium pH gradient electrophoresis. Much less diversity was observed in the major histocompatability molecule expression of the African green monkeys of Barbados than in the African cohort.

Stage-specific remodeling of the mammary gland basement membrane during lactogenic development.

Several in vitro studies have highlighted the importance of extracellular matrix proteoglycans as regulators of mammary epithelial cell differentiation. In this study monospecific antibodies to heparan (HS) and chondroitin/dermatan sulfate (CS/DS) proteoglycans were used to determine their possible significance in vivo. Immunohistochemical studies of proteoglycan distribution in virgin, pregnant, and lactating tissue demonstrated that heparan sulfate proteoglycan was found in the epithelial cell basement membrane at all stages of development. This was also true for CS/DS proteoglycans, but the relative proportions of CS and DS were strikingly modulated during lactogenesis. Notably DS glycosaminoglycans were the dominant components of the basement membrane in lactating tissue but CS glycosaminoglycans were the major proteoglycans of pregnant tissue. The possible significance of these results for gene expression in the gland is discussed.

Contribution of disulfide bonds in the carboxyl terminus of the human immunodeficiency virus type I gp120 glycoprotein to CD4 binding.

The carboxyl half of the HIV-1 gp120 glycoprotein, which has been implicated in binding to the CD4 receptor, contains two disulfide bonds linking cysteine residues 378-445 and 385-418. To examine the necessity of these disulfide bonds for the formation and/or maintenance of a gp120 glycoprotein competent for CD4 binding, we created mutants of a soluble form of gp120 in which combinations of these cysteine residues were altered. The mutant glycoproteins were examined for export from the expressing cell and for CD4 binding ability. Mutant gp120 molecules lacking both disulfide bonds were not stably expressed or exported. However, mutants for which either disulfide bond could form were exported and were fully competent for CD4 binding. In some cases, the presence of one of the pair of linked cysteines exerted more detrimental effects on export or CD4 binding than did alteration of both cysteines. Thus, the evaluation or the contribution of a particular disulfide bond to a phenotype should include studies in which both cysteines involved in the bond are simultaneously altered.

cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences.

A 2.1-kilobase cDNA coding for a surface protein of mammary epithelial cells has been isolated from a mouse mammary gland lambda gt11 cDNA library. Sequence analysis of this cDNA reveals an open reading frame of 1389 base pairs that defines a protein with a molecular mass of 51.5 kDa. Structural analysis of the predicted sequence identifies two putative functional domains of the protein: (i) an N-terminal cysteine-rich region that is similar to epidermal growth factor-like domains of Drosophila Notch-1 protein and (ii) a large segment of the sequence that exhibited 54.5% identity with C-terminal domains of human coagulation factors VIII and V. These similarities in structure are used to predict the possible functions of the protein and its means of interaction with the cell surface. mRNA expression was detectable in mammary tissue from nonpregnant animals but was maximal in the lactating gland. In cultured cells, mRNA levels also correlated with the degree of cellular differentiation.