Postnatal pre- and postexposure passive immunization strategies: protection of neonatal macaques against oral simian-human immunodeficiency virus challenge.

Simian-human immunodeficiency viruses (SHIV) allow the evaluation of antiviral strategies that target the envelope glycoproteins of the human immunodeficiency virus 1 (HIV-1) in macaques. We previously protected neonates from oral challenge with cell-free SHIV-vpu+ by passive immunization with synergistic human neutralizing monoclonal antibodies (mAbs) (Baba et al., Nat Med 6:200-206, 2000). mAbs were administered prenatally to pregnant dams and postnatally to the neonates. Here, we used solely postnatal or postexposure mAb treatment, thus significantly reducing the amount of mAbs necessary. All neonatal monkeys were also protected with these abbreviated mAb regimens. Our results are directly relevant for humans because we used mAbs that target HIV-1 envelope glycoproteins. Thus, the large-scale use of passive immunization with neutralizing mAbs may be feasible in human neonates. The mAbs, being natural human proteins, can be expected to have low toxicity. Passive immunization has promise to prevent intrapartum as well as milk-borne virus transmission from HIV-1-infected women to their infants.

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.

Passive immunization against oral AIDS virus transmission: an approach to prevent mother-to-infant HIV-1 transmission?

To develop immunoprophylaxis regimens against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, we established a simian-human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al: AIDS Res Hum Retroviruses 10:351-357, 1994). We protected four neonates from oral SHIV-vpu+ challenge by ante- and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti-HIV-1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann-Lehmann et al: Nature Med 6:200-206, 2000), which recognize the CD4-binding site of Env, a glycosylation-dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV-vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb-treated neonates was protected from infection and two maintained normal CD4+ T-cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV-1 transmission in humans.

Potent neutralization of primary human immunodeficiency virus clade C isolates with a synergistic combination of human monoclonal antibodies raised against clade B.

OBJECTIVES: We investigated the ability of several human neutralizing monoclonal antibodies (mAbs), originally raised against human immunodeficiency virus (HIV) clade B isolates, to neutralize primary clade C isolates as single agents and in combination. STUDY DESIGN/METHODS: HIV clade C isolates from five different countries were tested for susceptibility to neutralization by anti-clade B mAbs in human peripheral blood mononuclear cells. Monoclonal antibody combinations were evaluated for possible synergy. RESULTS: All 20 primary HIV clade C isolates could be neutralized 97.5% to 100% by a quadruple combination of mAbs IgG1b12, 2G12, 2F5, and 4E10. These mAbs recognized conserved epitopes and were highly synergistic, resulting in strong cross-clade neutralization. CONCLUSIONS: In our previous experiment, a synergistic combination of human neutralizing mAbs protected all macaque neonates against oral challenge with a simian-human immunodeficiency virus encoding HIV env. Together, our data suggest that passive immunization with currently available anti-clade B mAbs could play a role in preventing HIV clade C transmission through breastfeeding.

Postnatal passive immunization of neonatal macaques with a triple combination of human monoclonal antibodies against oral simian-human immunodeficiency virus challenge.

To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351-357, 1994). Using this model, neonates were protected from mucosal SHIV-vpu(+) challenge by pre- and postnatal treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and 2F5 (Baba et al., Nat. Med. 6:200-206, 2000). In the present study, we used this MAb combination only postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu(+) challenge, while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination against the more pathogenic SHIV89.6P, a chimeric virus encoding env of the primary HIV89.6. Remarkably, the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4(+) T-cell decline. In contrast, all control animals had dramatic drops in their CD4(+) T cells by 2 weeks postexposure. We conclude that our triple MAb combination partially protected against mucosal challenge with the highly pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1.

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.

A human anti-HIV autoantibody enhances EBV transformation and HIV infection.

A highly specific, human IgG mAb, F223, which reacts with both HIV-1-infected cells and uninfected lymphoid cells, has been derived. F223 reacts with gp120 but fails to neutralize viral infection. The antibody does enhance HIV-1 infection in a complement-dependent manner. The autoantigen recognized by F223 is expressed on a small percentage of T cells and NK cells and the majority of B cells. Immunoprecipitation demonstrates F223 reactivity with an as of yet unidentified 159-kDa protein in uninfected lymphoid cells. This reactivity with uninfected cells is inhibited by free gp120 demonstrating the cross-reactive nature of this antibody. The F223 light chain demonstrates strong homology to VLlambda2 family genes whereas the heavy chain is most homologous (84%) to the germline gene VH3-H.11. In vivo usage of VH3 family genes by F223 and an anti-HIV-1 (gp41) human mAb, 3D6, with related autoreactivity, suggests that VH3 sequences may be important components of potentially pathogenic human anti-HIV-1 envelope autoantibodies. F223 was isolated from an HIV-1 infected individual with lymphoma and in vitro F223 significantly enhances EBV transformation of normal B cells and increases immunoglobulin production without affecting B cell proliferation. Characterization of this antibody response may provide important insights and mechanistic information on HIV pathogenesis.

Minimal incidence of serum antibodies reactive with intact primary isolate virions in human immunodeficiency virus type 1-infected individuals.

Immunoglobulin G reactive with primary isolate virions was detected in 36% of serum samples from individuals infected with human immunodeficiency virus type 1. Of these individuals, serum samples from only 7% captured significant quantities of virus. Virion-specific antibody correlated with CD4 counts and, of more significance, primary isolate neutralization. Further dissection of this response should lead to the identification of antibodies and antigenic epitopes for vaccine purposes.

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.

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.

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.

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.

Influence of heavy chain constant regions on antigen binding and HIV-1 neutralization by a human monoclonal antibody.

F105, a neutralizing IgG1 kappa human mAb, is reactive with a discontinuous epitope within the gp120 CD4 binding site. Because isotype usage may affect Ab function, we examined the effect of isotype on Ag/Ab interactions and HIV-1 neutralization. An IgG3 kappa Ab was prepared by linking the variable regions of F105 to cloned human kappa and gamma 3 constant regions. Immunoreactivity of F105 IgG1 and IgG3 with IIIB-, MN-, and RF-infected cells was equivalent. Inhibition of binding and fusion of IIIB to uninfected cells and neutralization of IIIB virus was comparable for F105 IgG1 and IgG3, with 14 to 23 micrograms/ml required for 90% neutralization. In contrast, F105 IgG3 was marginally more effective at inhibition of MN binding/fusion and significantly more effective at neutralization of MN virus (62 micrograms/ml for IgG3 and > 100 micrograms/ml for IgG1 to achieve 90% neutralization). Despite high affinity binding to RF-infected cells, F105 IgG1 minimally neutralizes free RF virus. F105 IgG3 is dramatically more effective against the RF isolate, with 2 to 20 micrograms/ml of Ab required for 50% neutralization. Both isotypes were relatively ineffective at inhibition of RF binding/fusion. Thus, whereas affinity with native Ags on the surface of HIV-1-infected cells was unaffected by heavy chain constant regions, Ab isotype can strongly influence virion neutralization. Structural changes in gp120, as a result of increased flexibility conferred by the elongated IgG3 hinge region, are suggested as a possible mechanism to increase neutralization of selected HIV-1 isolates. These results may have significant implications in the design of immunotherapeutic and vaccine agents.

Plasma pharmacokinetics and biological activity of a human immunodeficiency virus type 1 neutralizing human monoclonal antibody, F105, in cynomolgus monkeys.

The IgG1 kappa human monoclonal antibody (HMab), F105 reacts with a discontinuous epitope on the CD4 binding site (CD4BS) of human immunodeficiency virus type 1 (HIV-1)/gp120 and has broad neutralizing activity. F105 HMab (60 mg/kg bolus) was administered intravenously to four monkeys and serum was collected at intervals to determine pharmacokinetics in a primate model. Average serum F105 concentrations, as determined by enzyme-linked immunosorbent assay, were analyzed with MINSQ software using a two-compartment, first-order model. The half-life for the alpha phase of the distribution curve is 6.7 h and for the beta elimination phase, 9.6 days. The volume of distribution is 0.65 L/kg and the rate of clearance 2 ml/kg/h. Serum levels of 1.3-1.6 mg/ml of F105 were maintained for 24 h. When monkey serum from day 15 postdose was tested, total serum F105 was 230 +/- 79 micrograms/ml and was immunoreactive with cells infected with the MN and IIIB strains of HIV-1 as determined by flow cytometry. Binding activity was identical to that obtained with stock F105 HMab. Identical neutralizing activity between the injected and uninjected antibody was also observed. Thus, serum neutralizing titers (90%) of 1:2000 at peak and 1:30 at day 15 postdose for MN virus were observed. These data indicate that high in vivo levels of HMab F105 can be attained by single bolus administration with full retention of biological activity. Of importance, levels of antibody necessary for effective neutralization can be achieved and maintained.

Effect of antibody valency on interaction with cell-surface expressed HIV-1 and viral neutralization.

F(ab) and F(ab')2 fragments of the human mAb, F105, were compared to intact IgG1 for binding to the CD4 binding site of HIV-1/gp120 on the surface of infected cells and viral neutralization. F105 IgG1 and F(ab')2 bound to IIIB, MN, and RF infected cells and neutralized these strains in an identical fashion, whereas strain-specific differences were observed in F(ab) activity. Although F105 F(ab) bound with equivalent affinity to IIIB-infected cells, there was a 4- to 10-fold decrease in the neutralization of IIIB by monovalent F(ab) compared to the bivalent molecules. F105 F(ab) demonstrated both diminished binding and neutralization of the MN strain and failed to bind or neutralize the RF strain. When cooperativity of V3 loop antibody (V3ab) with F105 IgG and fragments was examined, the binding of F105 IgG and F(ab')2 to IIIB-, MN-, or RF-infected cells was modestly enhanced by V3ab; viral neutralization was substantially enhanced by the combination of V3ab and F105 IgG and F(ab')2. The combination of F105 F(ab) with V3ab also resulted in significant cooperative neutralization of IIIB and MN, but the lack of F105 F(ab) binding and neutralization of RF was unaltered by V3ab. These results suggest that bivalent interaction may be important in binding and neutralization of virus, and support the notion that this interaction may depend on conformational changes in oligomeric gp120 on intact virions and cell surface rather than on affinity or steric effects.

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.

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.

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.

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.

Effect of immunosuppressive therapy on cytolytic activity of immunodeficient mice: implications for xenogeneic transplantation.

Despite major deficits in their immune system, SCID, Nude, and NIH III mice reject allo- and xenografts, particularly leukemic cell lines, albeit less readily than immunologically intact mice. Since variation among these immunodeficient mouse strains in rejection of a human lymphoid cell line (CCRF-CEM) parallels splenic non-MHC-mediated cytolytic activity, non-MHC-restricted cytolytic activity may be responsible for retained resistance to leukemic cell transplantation. SCID mice that had the least cytolytic activity accepted 100% of their grafts. The converse was true for NIH III mice that showed the greatest cytolytic activity and were relatively resistant to CEM cell engraftment. Different approaches to ablate NK activity and thus enhance engraftment led to variable results for each strain. A single dose (500 micrograms) of anti-asialoGM1 (AsGM1) markedly reduced NK activity in SCID and NIH III mice by 60 and 40%, respectively. A moderate 20% decrease was seen in Nude mice at this dose. In contrast, gamma irradiation suppressed NK activity by greater than 80% of baseline levels in all three strains. Of importance, total cytolytic activity in immunosuppressed Nude and NIH III mice, although significantly depressed compared to untreated mice of the same strain, still remained higher than that seen in nonimmunosuppressed SCID mice. Enhanced engraftment and systemic dissemination of CEM cells in immunosuppressed mice correlated directly with decreased total splenic cytolytic activity in all three strains. These results have implications for the use of immunodeficient models for transplantation, tumor immunobiology, and engraftment of a human immune system.