Virus entry via the alternative coreceptors CCR3 and FPRL1 differs by human immunodeficiency virus type 1 subtype.

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.

HIV type 1 subtype E-infected patients with broadened, dual (B/E) V3 loop serology have increased cross-neutralizing antibodies.

The two prevalent subtypes of HIV-1 circulating in Thailand are subtypes E and B. While the most prevalent subtype continues to be E using molecular typing assays, immunologically, a subset of subtype E-infected patients (3.4% in 1997) have binding antibodies to both the E and B V3 loops in a peptide ELISA. To assess the potential function of this dual (B/E) V3 reactivity, plasmas from patients with genetically defined HIV-1 subtype E infection and either E or B/E V3 serotypes were compared for magnitude and breadth of neutralization of seven primary and laboratory-adapted subtype B and E viruses. Dually reactive (B/E) plasmas showed significantly increased cross-neutralizing activity against subtype B viruses (p < 0.001), and increased neutralization of the panel of viruses overall (p < 0.02), as compared to monoreactive E serotype plasmas. While the total envelope binding antibody titers to both subtype B and E envelopes did not differ significantly between the E and B/E plasmas, 67% of B/E plasmas neutralized >50% of the viruses in the panel, and only 14% of E plasmas showed this broadened neutralizing activity. These data suggest that dual (B/E) V3 loop reactivity may be a marker of broader immune recognition of HIV envelope epitopes in subtype E-infected patients. V3 loop antibody, perhaps in conjunction with antibodies to additional epitopes, may play a role in neutralization of virus isolates from Thailand.

Construction and biological characterization of infectious molecular clones of HIV-1 subtypes B and E (CRF01_AE) generated by the polymerase chain reaction.

We previously described the use of extended polymerase chain reaction (PCR) to amplify contiguous 9.2-kilobase (kb) single-long terminal repeat (LTR) proviral sequences from HIV-1 genetic subtypes A through G. We now extend these findings by describing a novel vector system to recover infectious molecular clones from long PCR amplicons. Directional ligation of 9.2-kb proviral amplicons into a recovery vector reconstitutes missing LTR sequences, providing candidate molecular clones for infectivity screening. We show that a previously characterized infectious molecular clone of HIV-1 retains its biological properties upon recovery with this strategy. Three additional infectious molecular clones generated, from primary isolates of subtype B (HIV-1(WR27)) and circulating recombinant form 01_AE (subtype E) (HIV-1(CM235)) by subtype-specific LTR reconstitution, displayed biological properties reflecting their cognate parental isolates. This represents the first report of infectious molecular clones from circulating recombinant form 01_AE (subtype E).

A flow cytometric method for measuring neutralization of HIV-1 subtype B and E primary isolates.

BACKGROUND: Clinical trials testing candidate human immunodeficiency virus type 1 (HIV-1) vaccines have required the use of HIV neutralization assays to detect responses to specific geographic subtypes of HIV-1. The variability in results seen with current p24 neutralization assay endpoints prompted us to assess the utility of flow cytometry for monitoring the neutralization of HIV-1 primary isolates. METHODS: A modified neutralization assay was performed using CD8-depleted peripheral blood mononuclear cells (PBMC). The cells were fixed, permeabilized, stained with a directly conjugated HIV-1 p24 monoclonal antibody, and analyzed by flow cytometry. HIV-1 subtype B' and E primary isolates were tested using pooled sera or plasma from subtype B' or E infected patients. RESULTS: Primary isolate cultures (without neutralizing antibody) showed from 18% to 42% p24(+) cells, depending on the virus. Less than 0.2% p24(+) cells were detected in uninfected cultures. Subtype-specific neutralization of viruses was observed using plasma or serum pools; neutralization ranged from 0% to 99% reduction of infected cells. CONCLUSIONS: Flow cytometric detection of intracellular HIV-1 p24 can be used as an endpoint assay to assess neutralization of HIV-1 subtypes B' and E primary isolates. This enumerative method has the advantage of identifying intracellular p24 in specific subsets at an early culture timepoint. It also provides an alternative quantitative endpoint for HIV neutralization assays.

Differential role of V3-specific antibodies in neutralization assays involving primary and laboratory-adapted isolates of HIV type 1.

To identify epitopes important in neutralizing primary HIV-1 isolates, we have selectively depleted HIV-1 sera of antibodies specific for the third hypervariable region (V3) of the HIV-1 envelope glycoprotein gp120, and then assessed the functional consequences of such depletion in neutralization assays. The nucleotide sequence of the V3 loop region from HIV-1 PBMC DNA was determined for three HIV-1-infected patients, corresponding peptides were synthesized, and then subsequently used for V3 depletion of the patient sera. Depletion using a single clade B V3 peptide was capable of depleting > 98% of binding antibodies to multiple clade B V3 peptides, including those with changes within the GPGX tip of the loop. Depleted and undepleted sera were studied for their ability to neutralize both laboratory-adapted HIV-1MN and two primary HIV-1 isolates with known V3 sequences, using a viral infectivity reduction assay. While the majority of HIV-1MN neutralization was lost on V3 depletion, the loss in neutralization capacity against primary isolates by these same V3-depleted sera was substantially less pronounced. This suggests that V3 peptide-specific antibodies within HIV-1 serum play a fundamentally different role in mediating neutralization in assays involving laboratory-adapted and primary isolates and implicates antibodies with epitope specificities outside of V3 as major determinants in neutralization assays involving primary isolates.

Dissociation rate of antibody-gp120 binding interactions is predictive of V3-mediated neutralization of HIV-1.

mAbs specific for the V3 loop of HIV-1 are capable of neutralizing laboratory strains of HIV-1 in vitro. In this report we use surface plasmon resonance and biosensor technology to demonstrate that the ability of V3-specific mAbs to neutralize HIV-1(MN) correlated with the dissociation rate constant of the homologous mAb-gp120 binding interaction. mAbs capable of binding diverse strains of gp120 with similar association rate constants exhibited marked differences in the dissociation rate. The dissociation rate, and not the association rate, was found to be predictive of the neutralization capacity of the mAb. Furthermore, synthetic peptides corresponding to the V3 loop of HIV-1(IIIB, MN) yielded quantitatively similar binding kinetic profiles abrogating the need for purified recombinant gp120 protein and potentially facilitating the screening of more diverse isolates. Biosensor immobilized V3 peptides were found to mimic their conformational structure in solution. This offers advantages to peptides studied by ELISA where some degree of denaturation and masking of epitopes can occur upon adsorption of peptides to plastic surfaces. The impact of amino acid substitutions within epitopes on subsequent mAb binding was dissected by observing alterations in dissociation rates. The technique provides rapid kinetic analyses of V3 Ab binding interactions with diverse V3 sequences, facilitating the efficient screening and selection of those most likely to possess the broadest and most potent HIV-1 neutralizing potentials.

Characterization of substance P binding to human monocytes/macrophages.

Substance P (SP), a member of the tachykinin family of neuropeptides, can immunomodulate human T cells and monocytes. SP has been shown to stimulate human monocytes to produce inflammatory cytokines and superoxide ions, and it enhances tumoricidal activity in vitro. A specific SP receptor, however, has not been identified on human monocytes/macrophages. In this study, we report that 125I-SP binds to human monocytes/macrophages with high affinity and specificity (Kd = 2.7 x 10(-8) to 5.5 x 10(-8) M). Our measurements of binding affinity to this single class of receptors were possible only when experiments were performed in the presence of excess serine proteinase inhibitor (serpin) enzyme complex receptor ligand. We determined that 125I-SP bound to a specific receptor on human monocytes/macrophages and that this binding was detectable as early as 6 h and was maintained throughout 6 to 8 weeks in culture. Modulation of the diverse immunological and inflammatory effects of SP on human monocytes may be mediated through this specific SP receptor.

Serum IgA-mediated neutralization of HIV type 1.

The sera of 33 HIV-1-infected individuals, previously shown to neutralize HIV-1MN in vitro, were screened by ELISA for IgA reactivity against rgp120MN and a synthetic V3MN loop peptide. Six were selected for evaluation of the effect of serum IgA from infected individuals on the in vitro infection of susceptible target cells by HIV-1MN. By using protein G immobilized on Sepharose, we depleted the sera of IgG to a level undetectable by nephelometry and viral envelope-specific ELISA. The IgA component of the IgG-depleted serum was affinity purified with immobilized jacalin, a lectin that selectively binds the IgA1 fraction of human Ig. IgG-depleted sera and purified IgA1 serum fractions showing IgA reactivity against rgp120MN and V3MN by ELISA inhibited the in vitro infection of CEM-ss cells by HIV-1MN, but sera depleted of both IgG and IgA1 did not. These data show that, like serum IgG, serum IgA from selected HIV-1-infected individuals is capable of neutralizing HIV-1MN in vitro. The biologic significance of this observation and the identities of serum IgA-recognized HIV-1 neutralization epitopes remain to be determined.

Anoxia induces human immunodeficiency virus expression in infected T cell lines.

The effects of oxygen deprivation, or anoxia, on human immunodeficiency virus (HIV-1) expression in chronically (ACH.2) and acutely (H9/HIV-1-IIIB) infected cell lines was investigated. Temporary cellular anoxia has previously been shown to activate transcription of endogenous type C leukemia virus sequences, resulting in a significant increase in retroviral RNA within the cell (1). Here we report a 15-fold increase in HIV-1-specific RNA in unstimulated ACH.2 T cells within 24 h of anoxia. This induction of RNA is accompanied by an accumulation of intracellular p24 gag protein as well as an increase in envelope protein. Anoxia induces a further increase in total HIV-1 RNA in ACH.2 cells prestimulated to produce virus by phorbol 12-myristate 13-acetate and in H9 T cells acutely infected with HIV-1-IIIB. The induction of RNA in ACH.2 cells appears to be reversible. Anoxic culture for 24 h followed by a 24-h re-oxygenation period results in a return to "resting state" levels of HIV-1 RNA. These data indicate that oxygen tension within the cellular environment modulates HIV-1 expression, providing a model system in which to study the reversible regulation of HIV-1 RNA and viral gene products within the cell.

Two-dimensional polyacrylamide gel electrophoresis analysis of phosphorylated, membrane-localized ras p21 proteins.

The ras p21 oncogene product migrates as a heterogeneous series of polypeptides as resolved by both one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). We have prepared polyclonal rat serum antibody to ras p21 and used this as well as monoclonal antibodies to immunoprecipitate forms of p21 synthesized in vivo in transformed NIH3T3 cells. Two-dimensional PAGE of p21 resolved two distinct groups of polypeptides, one acidic (pI 4.8-5.3) which we call the "A" forms, and one less acidic or more basic (pI 6.5-7.0) which we call the "B" forms. It is the membrane-localized, B forms of v-ras-Ha p21 that are predominantly phosphorylated in vivo.

An unusual oxygen-sensitive lactate dehydrogenase isozyme associated with Kirsten murine sarcoma virus in human serum.

An unusual isozyme of lactate dehydrogenase, lactate dehydrogenase associated with Kirsten murine sarcoma virus (LDHk) was found in the sera of many patients with malignant tumors, while the sera of healthy persons had little or no such activity. This isozyme was detectable only when assayed in a nitrogen atmosphere, and its activity showed little or no relationship to the total lactate dehydrogenase activity as measured by a standard clinical assay. The activity of serum LDHk appeared to be correlated with the presence of known metastases. Increased serum LDHk appeared in a wide variety of patients with cancer, although it appeared to be more common in certain types of cancer. Increased serum LDHk activity was also found in the sera of some patients with nonmalignant disease. The activity of serum LDHk may be useful to monitor recurrence or response to therapy in certain types of cancer.