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Susan W Barnett

Publications and source records attributed to Susan W Barnett.

26 records · Page 2Linked to original sources
PubMed2003

Variability at human immunodeficiency virus type 1 subtype C protease cleavage sites: an indication of viral fitness?

Naturally occurring polymorphisms in the protease of human immunodeficiency virus type 1 (HIV-1) subtype C would be expected to lead to adaptive (compensatory) changes in protease cleavage sites. To test this hypothesis, we examined the prevalences and patterns of cleavage site polymorphisms in the Gag, Gag-Pol, and Nef cleavage sites of C compared to those in non-C subtypes. Codon-based maximum-likelihood methods were used to assess the natural selection and evolutionary history of individual cleavage sites. Seven cleavage sites (p17/p24, p24/p2, NC/p1, NC/TFP, PR/RT, RT/p66, and p66/IN) were well conserved over time and in all HIV-1 subtypes. One site (p1/p6(gag)) exhibited moderate variation, and four sites (p2/NC, TFP/p6(pol), p6(pol)/PR, and Nef) were highly variable, both within and between subtypes. Three of the variable sites are known to be major determinants of polyprotein processing and virion production. P2/NC controls the rate and order of cleavage, p6(gag) is an important phosphoprotein required for virion release, and TFP/p6(pol), a novel cleavage site in the transframe domain, influences the specificity of Gag-Pol processing and the activation of protease. Overall, 58.3% of the 12 HIV-1 cleavage sites were significantly more diverse in C than in B viruses. When analyzed as a single concatenated fragment of 360 bp, 96.0% of group M cleavage site sequences fell into subtype-specific phylogenetic clusters, suggesting that they coevolved with the virus. Natural variation at C cleavage sites may play an important role, not only in regulation of the viral cycle but also in disease progression and response to therapy.

Amino Acid Sequence↗
PubMed2003

Purification, characterization, and immunogenicity of a soluble trimeric envelope protein containing a partial deletion of the V2 loop derived from SF162, an R5-tropic human immunodeficiency virus type 1 isolate.

The envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the major target of neutralizing antibody responses and is likely to be a critical component of an effective vaccine against AIDS. Although monomeric HIV envelope subunit vaccines (gp120) have induced high-titer antibody responses and neutralizing antibodies against laboratory-adapted HIV-1 strains, they have failed to induce neutralizing antibodies against diverse heterologous primary HIV isolates. Most probably, the reason for this failure is that the antigenic structure(s) of these previously used immunogens does not mimic that of the functional HIV envelope, which is a trimer, and thus these immunogens do not elicit high titers of relevant functional antibodies. We recently reported that an Env glycoprotein immunogen (o-gp140SF162DeltaV2) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162, when used in a DNA priming-protein boosting vaccine regimen in rhesus macaques, induced neutralizing antibodies against heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIV(SF162P4) virus. Here we describe the purification of this protein to homogeneity, its characterization as trimer, and its ability to induce primary isolate-neutralizing responses in rhesus macaques. Optimal mutations in the primary and secondary protease cleavage sites of the env gene were identified that resulted in the stable secretion of a trimeric Env glycoprotein in mammalian cell cultures. We determined the molecular mass and hydrodynamic radius (R(h)) using a triple detector analysis (TDA) system. The molecular mass of the oligomer was found to be 324 kDa, close to the expected M(w) of a HIV envelope trimer protein (330 kDa), and the hydrodynamic radius was 7.27 nm. Negative staining electron microscopy of o-gp140SF162DeltaV2 showed that it is a trimer with considerable structural flexibility and supported the data obtained by TDA. The structural integrity of the purified trimeric protein was also confirmed by determinations of its ability to bind the HIV receptor, CD4, and its ability to bind a panel of well-characterized neutralizing monoclonal antibodies. No deleterious effect of V2 loop deletion was observed on the structure and conformation of the protein, and several critical neutralization epitopes were preserved and well exposed on the purified o-gp140SF162DeltaV2 protein. In an intranasal priming and intramuscular boosting regimen, this protein induced high titers of functional antibodies, which neutralized the vaccine strain, i.e., SF162. These results highlight a potential role for the trimeric o-gp140SF162DeltaV2 Env immunogen in a successful HIV vaccine.

Amino Acid Sequence↗
PubMed2003

Changes in the immunogenic properties of soluble gp140 human immunodeficiency virus envelope constructs upon partial deletion of the second hypervariable region.

Immunization of macaques with the soluble oligomeric gp140 form of the SF162 envelope (SF162gp140) or with an SF162gp140-derived construct lacking the central region of the V2 loop (DeltaV2gp140) results in the generation of high titers of antibodies capable of neutralizing the homologous human immunodeficiency virus type 1 (HIV-1), SF162 virus (Barnett et al. J. Virol. 75:5526-5540, 2001). However, the DeltaV2gp140 immunogen is more effective than the SF162gp140 immunogen in eliciting the generation of antibodies capable of neutralizing heterologous HIV-1 isolates. This indicates that deletion of the V2 loop alters the immunogenicity of the SF162gp140 protein. The present studies were aimed at identifying the envelope regions whose immunogenicity is altered following V2 loop deletion. We report that the antibodies elicited by the SF162gp140 immunogen recognize elements of the V1, V2, and V3 loops, the CD4-binding site, and the C1 and C2 regions on the homologous SF162 gp120. With the exception of the V1 and V2 loops, the same regions are recognized on heterologous gp120 proteins. Surprisingly, although a minority of the SF162gp140-elicited antibodies target the V3 loop on the homologous gp120, the majority of the antibodies elicited by this immunogen that are capable of binding to the heterologous gp120s tested recognize their V3 loops. Deletion of the V2 loop has two effects. First, it alters the immunogenicity of the V3 and V1 loops, and second, it renders the C5 region immunogenic. Although deletion of the V2 loop does not result in an increase in the immunogenicity of the CD4-binding site per se, the relative ratio of anti-CD4-binding site to anti-V3 loop antibodies that bind to the heterologous gp120s tested is higher in sera collected from the DeltaV2gp140-immunized animals than in the SF162gp140-immunized animals. Overall, our studies indicate that it is possible to alter the immunogenic structure of the HIV envelope by introducing specific modifications.

AIDS Vaccines↗
PubMed2002

Novel evolutionary analyses of full-length HIV type 1 subtype C molecular clones from Cape Town, South Africa.

Understanding the origin, distribution, and evolving dominance of HIV-1 subtype C strains is an important component in the design and evaluation of a globally effective AIDS vaccine. To better understand subtype C viruses, we constructed complete molecular clones of primary, CCR-5-using isolates from South Africa and analyzed the molecular phylogenies of these clones using best fitting evolutionary substitution models. Analyses were performed on three full-length sequences, and on the individual genes. All clones were nonrecombinant, and although two of three had open reading frames and intact splice sites, they were not infectious. At the genomic level, the models demonstrated the increasing variability of subtype C in South Africa. At the subgenomic level, they revealed marked differences in the evolutionary patterns of individual genes, a finding that suggests that the genes are under different selective pressures and constraints. These data underscore the dynamic nature of the subtype C epidemic and emphasize the need for continuous monitoring of local strains.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Species tropism of chimeric SHIV clones containing HIV-1 subtype-A and subtype-E envelope genes.

To analyze HIV-1 genes in a nonhuman primate model for lentivirus infection and AIDS, recombinant SIV/HIV-1 (SHIV) clones were constructed from two HIV-1 subtype-A isolates (HIV-1(SF170) and HIV-1(Q23-17) from individuals in Africa) and two HIV-1 subtype-E isolates (HIV-1(9466) and HIV-1(CAR402) from AIDS patients in Thailand and Africa), respectively. These four SHIV clones, designated SHIV-A-170, SHIV-A-Q23, SHIV-9466.33, and SHIV-E-CAR, contain envelope (env) genes from the subtype-A or -E viruses. Interestingly, SHIV-A-170, SHIV-A-Q23, and SHIV-9466.33 were restricted for replication in cultures of macaque lymphoid cells, whereas SHIV-E-CAR replicated efficiently in these cells. Additional studies to define the block to replication in macaque cells were focused on the subtype-E clone SHIV-9466.33. A SHIV intragenic env clone, containing sequence-encompassing V1/V2 regions of HIV-1(CAR402) and V3/V4/V5 regions of SHIV-9466.33, infected and replicated in macaque lymphoid cells. These results indicated that the sequence-encompassing V1/V2 region of HIV-1(9466) was responsible for the block of the SHIV-9466.33 replication in macaque cells. Analysis of viral DNA in acutely infected macaque cells revealed that SHIV-9466.33 was blocked at a step at/or before viral DNA synthesis, presumably during the process of virion entry into cells. In a fluorescence-based cell-cell fusion assay, fusion pore formation readily took place in cocultures of cells expressing the SHIV-9466.33 env glycoprotein with macaque T-lymphoid cells. Taken together, these results demonstrated that the block of SHIV-9466.33 replication in macaque cells is at an early step after fusion pore formation but before reverse transcription.

Animals↗
PubMed2002

Characterization of the South African HIV type 1 subtype C complete 5' long terminal repeat, nef, and regulatory genes.

Human immunodeficiency virus type 1 (HIV-1) subtype C has become the major etiological agent in the global and especially African epidemic. To gain better understanding of the genetic diversity and rapid transmission of HIV-1 subtype C, we have characterized the complete 5' long terminal repeat (LTR) region along with the regulatory genes tat and rev as well as the accessory gene nef of 14 South African HIV-1 subtype C isolates. Phylogenetic analysis revealed a subtype C 5' LTR cluster, as well as subclustering of our nef sequences with various subtype C strains separate from the India and China subclusters. At least 3 NF-kappaB sites were present in the 5' LTR of most isolates and 13 isolates had the subtype C-specific Rev truncation. Some length variation in exon 2 and the absence of a critical cysteine were found in Tat. Residue variation in the myristoylation signal and motifs involved in CD4 and MHC-I downregulation was recorded in our nef gene sequences.

Africa, Southern↗
PubMed2002

Purification and characterization of oligomeric envelope glycoprotein from a primary R5 subtype B human immunodeficiency virus.

Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1(US4). We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (R(h)) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller R(h) of the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.

AIDS Vaccines↗