Epstein-Barr virus-driven gene therapy for EBV-related lymphomas.

Genetic alterations in malignant tissues are potential targets for gene-based cancer therapies. Alternatively, aberrant expression of certain specific genes associated with malignant transformation may be envisioned to enhance the expression of chemosensitizing drugs. Epstein-Barr virus (EBV)-related B-cell lymphomas are fatal complications of immunosuppression due to AIDS, organ transplantation or congenital immune abnormalities. The malignant cells latently infected with EBV typically express the transcription factor EBNA2 as one of nine latent viral genes. We tested whether an EBNA2-responsive EBV promoter may selectively target EBV-related lymphoma cells by virus-regulated expression of a suicide gene. Using the BamC promoter driving a hygromycin-thymidine kinase fusion gene or controls, we demonstrated that sensitivity to ganciclovir was selectively enhanced in cells expressing EBNA2. Further, there was complete macroscopic regression of established B-cell lymphomas in mice with severe combined immunodeficiency disease (SCID mice) treated with a single course of ganciclovir. These data provide in vitro and in vivo support for a model of exploiting the molecular basis of tumor development to enhance the specificity of gene therapy.

Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection.

Although maternal human immunodeficiency virus type 1 (HIV-1) transmission occurs during gestation, intrapartum and postpartum (by breast-feeding), 50-70% of all infected children seem to acquire HIV-1 shortly before or during delivery. Epidemiological evidence indicates that mucosal exposure is an important aspect of intrapartum HIV transmission. A simian immunodeficiency virus (SIV) macaque model has been developed that mimics the mucosal exposure that can occur during intrapartum HIV-1 transmission. To develop immunoprophylaxis against intrapartum HIV-1 transmission, we used SHIV-vpu+ (refs. 5,6), a chimeric simian-human virus that encodes the env gene of HIV-IIIB. Several combinations of human monoclonal antibodies against HIV-1 have been identified that neutralize SHIV-vpu+ completely in vitro through synergistic interaction. Here, we treated four pregnant macaques with a triple combination of the human IgG1 monoclonal antibodies F105, 2G12 and 2F5. All four macaques were protected against intravenous SHIV-vpu+ challenge after delivery. The infants received monoclonal antibodies after birth and were challenged orally with SHIV-vpu+ shortly thereafter. We found no evidence of infection in any infant during 6 months of follow-up. This demonstrates that IgG1 monoclonal antibodies protect against mucosal lentivirus challenge in neonates. We conclude that epitopes recognized by the three monoclonal antibodies are important determinants for achieving substantial protection, thus providing a rational basis for AIDS vaccine development.

Vertical Transmission of HIV-1. Correlation with maternal viral load and plasma levels of CD4 binding site anti-gp120 antibodies.

Almost all childhood HIV-1 is now acquired through vertical transmission. Identifying factors that affect the rate of transmission may lead to the initiation of specific preventive strategies. In this study, antibody levels against different neutralizing epitopes on the envelope glycoprotein of HIV-1 (gp120) were measured in HIV-1-infected pregnant women that either transmitted HIV-1 to their infants (18 women) or did not (29 women). Differences in levels of antibodies directed against the monomeric gp120 molecule and against the V3 loop region of gp120 were not significantly different between the two groups studied. However, significant differences were observed in the levels of CD4 binding site antibodies, as determined by the ability of diluted maternal plasma to inhibit binding of the CD4 binding site monoclonal antibody F105 (mAb F105) to monomeric gp120. In addition, more nontransmitting mothers had low viral load as defined by having two or more negative HIV-1 viral cultures during pregnancy compared with transmitters. This pilot study suggests that in addition to higher viral load, low levels of CD4 binding site antibodies correlate with increased risk of HIV-1 vertical transmission. Passive immunotherapy with broadly neutralizing CD4 binding site antibodies should be considered as a strategy to reduce this risk.

Characterization of the cDNA of a broadly reactive neutralizing human anti-gp120 monoclonal antibody.

The F105 mAb, identified in an HIV-1-infected individual, binds to a discontinuous epitope on the HIV-1 gp120 envelope glycoprotein, blocks the binding of gp120 to the CD4 viral receptor, and neutralizes a broad range of HIV-1 isolates. This study reports the primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the mAb F105. This IgG1k mAb uses a VH gene member of the VH4 gene family (V71-4) and is productively rearranged with a D-D fusion product of the dlr4 and da4 germline DH genes and the JH5 gene. This rearranged heavy chain gene expresses the VH4-HV2a idiotope, which is seen in human monoclonal IgM cold agglutinins. The F105 Vk appears to be derived from the Humvk325 germline gene and is rearranged with a Jk2 gene. For both chains, the mutational pattern in the rearranged VH and VL genes is indicative of an antigen-driven process. These studies show that production of a broadly neutralizing anti-HIV-1 antibody that recognizes determinants within the CD4 recognition site of the envelope glycoprotein is achieved by rearrangement of the V71-4 and Humvk325 germline variable region genes along with selected individual point mutations in the rearranged genes.

V3-specific neutralizing antibodies in sera from HIV-1 gp160-immunized volunteers block virus fusion and act synergistically with human monoclonal antibody to the conformation-dependent CD4 binding site of gp120. NIH-NIAID AIDS Vaccine Clinical Trials Network.

Sera from 11 volunteers immunized with a recombinant HIV-1 gp160-expressing vaccinia virus (HIVAC-1e; Oncogen/Bristol-Myers Squibb, Seattle, WA) and boosted with baculovirus-derived rgp160 (VaxSyn; MicroGeneSys, Inc., Meriden, CT) were evaluated for functional serum antibodies and their epitopes. Sera obtained prior to boosting had undetectable HIV-1-specific IgG and neutralizing activity, and did not block HIV-1 from binding or fusing to CD4+ MT-2 cells. 14 d after boosting, sera from each volunteer contained HIV-1-specific IgG titers of 1:40 to 1:1,280. Five of these sera also contained neutralizing antibodies, where most or all neutralizing activity was blocked by a synthetic peptide corresponding to amino acids 307-330 of the V3 loop of gp120, indicating that neutralizing antibodies were mostly V3 loop-specific. All sera obtained after boosting contained HIV-1 binding/fusion-inhibition antibodies, and a significant portion of their activity was blocked by the V3 loop peptide, a result consistent with the presence of antibodies against the region of the V3 loop that participates in fusion. Three sera with V3 loop-specific neutralizing and fusion-inhibition antibodies were studied further. In competitive antibody binding experiments, antibodies reactive with the conformation-dependent, CD4 binding site of gp120 were undetectable in each serum. When evaluated in combination with a monoclonal antibody to the CD4 binding site of gp120, two sera demonstrated synergism in neutralizing assays, and all three sera demonstrated synergism in binding/fusion-inhibition assays, further indicating that the functional antibodies were primarily V3 loop-specific. The synergism also suggests that a vaccine that elicits strong serum antibody responses to both regions of gp120 may improve the potential for inducing protective immunity.

Surface membrane-expressed CD40 is present on tumor cells from squamous cell cancer of the head and neck in vitro and in vivo and regulates cell growth in tumor cell lines.

Because regional spread to lymph nodes without systemic spread is a relatively common event in squamous cell cancer of the head and neck (SCCHN), it is possible that lymphoid-related receptors or cytokines might directly impact the growth of these tumors. In the present study, we have shown by flow cytometry and Western blotting that the central lymphoid regulatory molecule, CD40, is expressed on the surface of all seven SCCHN tumor cell lines studied. Tumor cell lines also expressed epidermal growth factor (EGF) receptor, MHC class I, and CD95 (Fas) but did not uniformly express other important lymphoid regulatory molecules such as CD80, CD86, or interleukin (IL) 2 receptor components. CD40 ligation by trimeric CD40 ligand (CD40L) resulted in a 20-45% inhibition of tumor cell growth in three of seven cell lines tested. The cytokines IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-11, and IL-15 neither inhibited nor stimulated growth in any of the cell lines tested. EGF had pleiotropic effects on cell growth; it inhibited growth in two cell lines, stimulated growth in one cell line, and had no effect in four cell lines. When coligation by EGF and CD40L was studied, additive or supra-additive growth inhibition was seen in four cell lines. Three cell lines were unaffected by EGF, CD40, or coligation with both reagents. Examination of tumor tissues from 12 previously untreated patients representing a broad spectrum of patients presenting with SCCHN demonstrated CD40 expression in all 12 tumor specimens. This study supports the notion that CD40 is a regulatory molecule for the growth of SCCHN. The important role of CD40-CD40L interactions in the regulation of immune cells in the lymph node and the unique high-level expression of CD40L by these immune cells lend support to the hypothesis that this ligand/receptor pair is an important mediator of cell growth in SCCHN.

Pharmacokinetics of F105, a human monoclonal antibody, in persons infected with human immunodeficiency virus type 1.

F105 is a human monoclonal antibody that binds to the CD4 binding site of human immunodeficiency virus type 1 gp120 and neutralizes clinical and laboratory isolates of the human immunodeficiency virus. This phase I study investigated the disposition of the antibody in humans. F105 was administered over a 60-minute period at two dose levels, 100 and 500 mg/m2. Blood samples were obtained for up to 56 days. The clearance of the antibody was 0.33 ml/min with a corresponding half-life of approximately 13 days. Peak concentrations achieved at the higher dose level were 216.19 +/- 9.62 micrograms/ml. The disposition of the drug was linear for the doses studied. Simulations were performed to design future studies aimed at investigating the efficacy of the antibody. This study concluded that F105 can be administered as a bolus dose every 21 days.

Neutralization of HIV-1 by F105, a human monoclonal antibody to the CD4 binding site of gp120.

The functional ability of the human monoclonal antibody (HMab) F105 to neutralize commonly available laboratory strains and a selection of primary isolates of human immunodeficiency virus (HIV)-1 was studied. F105 is representative of a class of human antibodies that react with conformational epitopes within the discontinuous CD4 binding site on HIV-1 envelope glycoprotein gp120. F105 binds with relatively similar affinities to native antigen expressed on the surfaces of cells infected with each of five laboratory isolates tested (IIIB, SF2, MN, RF, and CC) and neutralizes SF2, IIIB, and MN with concentrations of antibody ranging from 140 ng to 10 micrograms/ml. Nonetheless, neutralization by F105 alone of RF and CC is poor at modest antibody concentrations despite high affinity binding to surface gp120 on infected cells. Neutralization of HIV-1 strains by F105 is unaffected by normal sera and cooperativity is observed with serum samples from HIV-1 infected patients. Of significance, neutralization of RF and MN by F105 is enhanced by some HIV-seropositive sera at low concentrations. F105 also neutralized 30% of HIV-1 primary isolates in lymphocyte cultures. Although it is unclear how relevant in vitro studies will be to in vivo events, these data allow comparison of F105 with other HMabs to the CD4 binding site and V3 loop and provide an in vitro reference for in vivo activity. These studies demonstrate that antibody interactions among different classes of antibodies may be important in in vivo neutralization of HIV-1.

Human monoclonal antibodies to the V3 loop of HIV-1 gp120 mediate variable and distinct effects on binding and viral neutralization by a human monoclonal antibody to the CD4 binding site.

Interactive effects between human monoclonal antibodies specific for the V3 loop (257-D and 447-D) and an epitope within the CD4 binding site (F105) of HIV-1 gp120 were evaluated for neutralization of viral cytopathogenicity and binding to HIV-infected cells. Regardless of antibody pair, only additive effects were observed in neutralization of MN and SF2 virus though each antibody alone had potent neutralizing activity on these strains. Significant cooperativity was observed between F105 and 447-D in neutralization of RF. Relatively high concentrations (> 100 micrograms/ml) of each individual antibody are required for partial neutralization (25--40%) of RF. Coincubation with 10 micrograms/ml of each antibody increased neutralization activity 3--4-fold more than predicted for additive effects alone. No enhancement was seen upon coincubation of F105 with 257-D which does not neutralize RF. Antibody interactions with native antigen on HIV-infected cells was measured by flow cytometry. Results were consistent with neutralization results in the majority of flow cytometry experiments; however, enhanced binding did not necessarily predict enhanced neutralization. These data support the notion that either a conformational change occurs with binding of V3 loop antibodies which enhances the binding and neutralizing activity of antibodies directed to the CD4 binding site of gp120 or vice versa, or new antigenic sites are exposed by the V3 loop antibodies on cell surfaces and virions. Of importance, cooperativity is observed even at very low antibody concentrations.

Loss of serum antibodies to a conformational epitope of HIV-1/gp120 identified by a human monoclonal antibody is associated with disease progression.

Serum antibody reactive with epitopes within the CD4 binding site (CD4BS) of HIV-1/gp120 on infected cells was measured by inhibition of binding of a human monoclonal antibody, F105, which recognizes a conformational epitope within this region. Serum samples from 27% of ARC/AIDS patients blocked binding of F105 to this epitope, while samples from 100% of asymptomatic seropositive patients blocked binding. F105 blocking activity increased in 87% of asymptomatic donors who maintained stable disease over a 3-6 year period and decreased in 50% of asymptomatic patients with progressive disease. Moreover, serum samples from patients with stable disease exhibited higher titers of F105 blocking activity. The presence of F105 blocking activity also correlated with serum neutralization of virus. When diluted 1:640, serum with low F105 blocking activity neutralized only 20-30% of viral cytopathic effect (CPE), while serum with high F105 blocking activity neutralized > 80%. Serum neutralization was enhanced by the addition of F105. Seroreactivity to infected cells was detectable within 6 months of seroconversion, but F105 blocking activity was delayed by an additional 6-12 months, as was the development of high titers of neutralizing antibody. These data support the notion that the humoral response to the CD4BS on gp120 may be important in the maintenance of health.

Cell-mediated immunity to the asexual blood stages of malarial parasites: animal models.

Studies using experimental models of malaria in immunodeficient mice and chickens have shown that resistance to blood-stage infection is mediated by protective antibodies and T cell-dependent cell-mediated mechanisms of immunity. Depending on the infecting species of Plasmodium and prior experience of the host, either humoral or cell-mediated immune mechanisms predominate. Cell-mediated immunity has been adoptively transferred with CD4+ splenic T cells, and with antigen-specific T cell lines and clones. Since ascending parasitemia occurs in all instances, the transferred cells do not kill plasmodia directly but appear to activate effector mechanisms capable of destroying the invading parasites. Both CD4+ and CD8+ T cells were found to increase in the spleens of malarious mice. Depletion of CD4+ T cells prevented nude mice adoptively transferred with immune splenic T cells from clearing parasitemia. In contrast, late treatment with anti-CD4 antibody had little if any effect. The converse was true when anti-CD8 antibody was utilized, i.e., a significant number of mice treated with anti-CD8 antibody after parasitemia became patent and had difficulty clearing blood parasites. These data suggest that during infection CD8+ T cells may become activated by CD4+ T cells responding to malarial antigens. These CD8+ T cells may be directly cytotoxic or secrete additional cytokines thereby amplifying the role of CD4+ T cells in the activation of anti-parasite effector mechanisms. Finally, a hypothesis is presented to explain how the parasite in natural infections may activate T cell-dependent effector mechanisms in order to control its numbers in host tissues thereby ensuring the survival of both parasite and host.

Functional activity of an HIV-1 neutralizing IgG human monoclonal antibody: ADCC and complement-mediated lysis.

The IgG1 kappa, human monoclonal antibody (HMAb), F105, was studied for functional activity in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). F105 reacts with a discontinuous epitope on the CD4 binding site of the HIV-1 envelope glycoprotein, gp120, expressed on the surfaces of infected cells and neutralizes diverse viral strains at antibody concentrations readily achievable in humans. Neither F105 nor serum (diluted 1:50) from HIV seropositive donors mediate CDC against an SF2-infected cell line with rabbit or human sera as a source of complement. F105 and HIV-1 sera mediate ADCC against the SF2 strain. Normal human serum reduced spontaneous lysis of SF2 by peripheral blood monocytes (PBM). Although mixing of F105 with normal human serum reduced the lysis observed (36 +/- 8 vs. 42 +/- 8%), this still was significantly greater than lysis in media (30 +/- 5%) or normal human serum (23 +/- 6%) (p less than .05). A murine antibody to CD16 significantly reduced spontaneous lysis observed with media (30 +/- 5 vs. 18 +/- 3%) while normal mouse serum had no effect (31 +/- 7%). ADCC mediated by F105 is completely abrogated by the anti-CD16 antibody (42 +/- 8 vs. 22 +/- 4%), while only a fraction of ADCC mediated by HIV sera is inhibited by anti-CD16 (60 +/- 9 vs. 46 +/- 6%), suggesting that several populations of effector cells function in ADCC mediated by the polyclonal sera. Thus, F105, as opposed to polyclonal sera, mediates ADCC through a CD16+ PBM population.

Phase I study of a human monoclonal antibody directed against the CD4-binding site of HIV type 1 glycoprotein 120.

A phase I dose escalation study was conducted with the human monoclonal anti-gp120 antibody F105, to evaluate the safety, pharmacokinetics, and functional activity of F105 in HIV-1-infected individuals. F105 is an IgG1(kappa) antibody reactive with a discontinuous epitope that overlaps the CD4-binding site of gp120. F105 neutralizes laboratory strains of HIV-1 and some primary isolates, and synergizes with other antibodies in neutralizing an expanded spectrum of isolates. Four patients each with CD4 counts between 200 and 500/mm3 received a single dose of F105 at 100 or 500 mg/m2, intravenously. Sustained levels of F105 were obtained in plasma, and there was no evidence of an immune response to F105 as determined by a double-antigen immunoassay. No patient experienced any toxicity. Infused antibody retained full functional activity as detected by the ability of sera to block the binding of labeled F105 to HIV-1-infected cells. Of note, all patients had preexisting antibody to the gp120 CD4-binding site. The ability to culture virus by quantitative microculture remained unchanged by this single dose of antibody. Thus, it can be concluded that F105 is safe and nontoxic as a single injection at the doses tested. Furthermore, the antibody retains full gp120-binding activity. In these patients, with preexisting CD4-binding site antibody, there is no evidence of anti-HIV-1 activity following a single antibody infusion.

Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41.

The immunoreactivity, functional activity, and molecular features of a human monoclonal antibody (HMAb), F240, from an HIV-1-infected individual have been studied. Flow cytometric analysis demonstrated that F240 is reactive with cells infected with a broad range of laboratory isolates but not with uninfected cells. Reactivity of F240 is greatly enhanced by preincubation of infected cells with soluble CD4, and to a much lesser extent, with F105, an HMAb reactive with the CD4-binding site of gp120. This enhancement is temperature dependent, with maximum enhancement observed at 37 degrees C, and suggests that the F240 epitope may be more accessible after gp120 has bound to CD4 in vivo. Immunoblot analysis reveals antigen specificity of F240 for gp41 or its precursor gp160. F240 specificity is mapped to the immunodominant region of the gp41 ectodomain by Pepscan analysis. This epitope has been implicated in eliciting nonprotective antibodies that enhance infection in the presence of complement. Consistent with this, F240 failed to neutralize laboratory isolates and enhanced viral infection in a complement-dependent manner. The F240 VH demonstrates extensive somatic mutations compared with the product of its closest homologous germline gene VH3-3.11. Most amino acid substitutions occur in CDR2, characteristic of an antigen-driven response, and in FR3, a phenomenon observed in other anti-HIV-1 envelope HMAbs. Primary structure analysis of the F240 heavy chain revealed strong homology in the CDR domains to an HMAb (3D6) reactive with the same gp41 region, which suggests that these HMAbs could define a potential human antibody clonotype.

Protection of neonatal macaques against experimental SHIV infection by human neutralizing monoclonal antibodies.

Neonatal macaques were completely protected against oral challenge with SHIV-vpu+, a simian-human immunodeficiency virus that encodes the envelope gene of a laboratory-adapted HIV strain, by pre- and post-natal treatment with a triple combination of human neutralizing monoclonal antibodies (mAbs). The mAbs were directed either against the CD4 binding site, a glycosylation-dependent gp120 epitope, or against a linear epitope on gp41. This triple combination was highly synergistic in vitro and neutralized primary HIV completely. Subsequently, oral challenge was performed with pathogenic SHIV89.6P, an animal-passaged variant of a chimeric virus that encodes the envelope gene of the primary, dual-tropic HIV89.6. Only post-natal treatment with a similar triple mAb combination was used. One out of 4 mAb-treated infants was completely protected from infection. In the other 3 treated animals, there was a tendency towards lower peak viral RNA loads compared with untreated controls. Two out of 4 mAb-treated infants maintained normal CD4+ T-cell numbers, in contrast to all controls that had steep declines at 2 weeks post-challenge. We conclude that the triple mAb combination significantly protected the neonates, even against mucosal challenge with pathogenic SHIV89.6P. Passively administered synergistic human mAbs may play a role in preventing mother-infant transmission of HIV, both against intrapartum transmission as well as against infection through breast milk. As passive immunization is a tool to assess correlates of immune protection, we conclude that the epitopes recognized by the mAbs in our combinations are important for AIDS vaccine development. Future passive immunization studies may reveal other important conserved epitopes.

Human antibody variable region gene usage in HIV-1 infection.

Human antibody variable region gene usage during human immunodeficiency virus type 1 (HIV-1) infection is examined in the following review, and several hypotheses are presented to account for the distinct patterns of antibody gene expression associated with infection. Evidence supporting qualitatively biased antibody gene expression has been derived from analysis of the human humoral immune response by isoelectric focusing (IEF) and serological and molecular studies of immunoglobulin (Ig) from different lymphoid compartments of HIV-1-infected patients. Preferential usage of heavy-chain variable region (VH) gene families 1 and 4 is supported by serological studies of serum Ig and molecular characterization of anti-HIV-1 human monoclonal antibodies derived from infected patients. Negative biases against VH3 family gene usage are detected by polymerase chain reaction (PCR) studies of peripheral blood lymphocytes from AIDS patients but not by combinatorial phage display library techniques. Biased antibody gene usage and expression during HIV-1 infection may be related to HIV-1 pathogenesis by limiting the available HIV-1 neutralizing repertoire. Further molecular characterization of anti-HIV-1 antibodies and in vivo expression of V-region genes during HIV-1 infection should provide important information regarding antibody gene expression and its relationship to HIV-1 pathogenesis.

T-cell immunity in murine malaria: adoptive transfer of resistance to Plasmodium chabaudi adami in nude mice with splenic T cells.

Acute infections caused by the murine malarial parasite Plasmodium chabaudi adami are resolved by antibody-independent mechanisms of immunity. The fact that athymic nude mice developed high-grade unrelenting malaria and died when infected with this parasite suggested a significant role for T lymphocytes. Using adoptive transfer techniques, we demonstrated that spleen cells from either nonimmune or immune donor BALB/c mice eventually suppressed P. chabaudi adami infections in histocompatible recipient nude mice in a dose-dependent manner. Infections in recipients of "immune" spleen cells were less severe, demonstrating a depressed peak parasitemia and a shortened duration of patent infection, than was observed in recipients of normal spleen cells. Also, when sufficient numbers of immune spleen cells were transferred, the second wave of parasitemia (characteristic of this infection in nonimmune mice) failed to occur. T lymphocytes mediated protection in recipient mice, since T-cell-enriched, but not B-cell-enriched, spleen cell fractions suppressed P. chabaudi adami infections in nude mice. Protection was best achieved with T cells that bore the L3T4 phenotype. Patent parasitemias developed in all recipient mice, suggesting that the grafted cells did not limit parasite growth directly but achieved this end by activating other as yet unidentified inhibiting cell systems.

Resolution of acute malarial infections by T cell-dependent non-antibody-mediated mechanisms of immunity.

While it is generally accepted that acute blood stage malarial infections are resolved through the actions of protective antibodies, we observed that resistance to acute infection with Plasmodium chabaudi adami was mediated by T cell-dependent cellular immune mechanisms independent of antibody. We now report that acute blood stage infections caused by three additional murine hemoprotozoan parasites, Plasmodium vinckei petteri, Plasmodium chabaudi chabaudi, and Babesia microti, appear to be controlled by similar T cell-dependent mechanisms of immunity. Mice rendered B cell deficient by lifelong treatment with goat anti-mouse immunoglobulin M (IgM) had IgM levels in serum of less than 0.6 micrograms/ml and contained precipitating amounts of goat anti-mouse IgM. When these B cell-deficient mice were infected with blood stage P. vinckei petteri, P. chabaudi chabaudi, or B. microti, they resolved their infections with kinetics similar to those seen in immunologically intact mice. Infected B cell-deficient mice did not produce antiparasite antibodies. As assayed by immunofluorescence, significant titers of parasite-specific antibody were present only in the sera of infected immunocompetent mice. In addition, only sera from infected immunocompetent mice immunoprecipitated metabolically labeled parasite antigens. In contrast to B cell-deficient mice, athymic nude mice failed to resolve acute P. vinckei petteri or B. microti infections. These data suggest that antibody-independent, T cell-mediated immune mechanisms play a more significant role in resisting acute blood stage infections caused by hemoprotozoa than was recognized previously.

Resistance to neutralization by broadly reactive antibodies to the human immunodeficiency virus type 1 gp120 glycoprotein conferred by a gp41 amino acid change.

A neutralization-resistant variant of human immunodeficiency virus type 1 (HIV-1) that emerged during in vitro propagation of the virus in the presence of neutralizing serum from an infected individual has been described. A threonine-for-alanine substitution at position 582 in the gp41 transmembrane envelope glycoprotein of the variant virus was responsible for the neutralization-resistant phenotype (M.S. Reitz, Jr., C. Wilson, C. Naugle, R. C. Gallo, and M. Robert-Guroff, Cell 54:57-63, 1988). The mutant virus also exhibited reduced sensitivity to neutralization by 30% of HIV-1-positive sera that neutralized the parental virus, suggesting that a significant fraction of the neutralizing activity within these sera can be affected by the amino acid change in gp41 (C. Wilson, M. S. Reitz, Jr., K. Aldrich, P. J. Klasse, J. Blomberg, R. C. Gallo, and M. Robert-Guroff, J. Virol. 64:3240-3248, 1990). It is shown here that the change of alanine 582 to threonine specifically confers resistance to neutralizing by antibodies directed against both groups of discontinuous, conserved epitopes related to the CD4 binding site on the gp120 exterior envelope glycoprotein. Only minor differences in binding of these antibodies to wild-type and mutant envelope glycoproteins were observed. Thus, the antigenic structure of gp120 can be subtly affected by an amino acid change in gp41, with important consequences for sensitivity to neutralization.