Human Anti-HIV-1 gp120 Monoclonal Antibodies with Neutralizing Activity Cloned from Humanized Mice Infected with HIV-1.

Broadly neutralizing, anti-HIV-1 gp120 mAbs have been isolated from infected individuals, and there is considerable interest in developing these reagents for Ab-based immunoprophylaxis and treatment. As a means to identify potentially new anti-HIV Abs, we exploited humanized NOD-scid IL2rγnull mice systemically infected with HIV-1 to generate a wide variety of Ag-specific human mAbs. The Abs were encoded by a diverse range of variable gene families and Ig classes, including IgA, and several showed significant levels of somatic mutation. Moreover, the isolated Abs not only bound target Ags with similar affinity as broadly neutralizing Abs, they also demonstrated neutralizing ability against multiple HIV-1 clades. The use of humanized mice will allow us to use our knowledge of HIV-1 gp120 structure and function, and the immune response targeting this protein, to generate native human prophylactic Abs to reduce the infection and spread of HIV-1.

Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition.

Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89) on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv) molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells.

Neutralization of HIV by milk expressed antibody.

BACKGROUND: In some areas of the world, mother-to-child transmission of HIV remains a significant problem in part due to widespread breastfeeding, which is essential because of scarce supply of a safe replacement, protection conferred by breast milk against many enteric illnesses, and cultural norms. We propose that sustained adequate levels of protective antibodies in breast milk will prevent transmission of HIV. METHODS: The HIV-neutralizing human monoclonal antibody b12 (IgG1) has been expressed as an IgA2 in CHO cells and shown to retain full immunoreactivity and neutralizing activity as the parental IgG1. The expression plasmids containing the b12 heavy and light chains were also used to construct milk-specific expression vectors using the GTC goat ß-casein expression vector to direct expression of linked genes to the mammary gland with subsequent secretion into the milk. Female transgenic mice were generated and following parturition, their milk was tested for antibody immunoreactivity with gp120 and neutralization of HIV. RESULTS: When milk-derived b12 IgA2 was compared with CHO-derived b12 IgA2 (or IgG1), immunoreactivity was retained. When tested for neutralization, milk-derived b12 IgA2 was at least comparable to CHO-derived antibody and in some cases, superior to CHO-derived antibody. Furthermore, milk that expressed b12 IgA2 was significantly more effective at mediating antibody-dependent cell killing. CONCLUSIONS: These results suggest that it is possible to achieve functional HIV-specific mAb in the milk of transgenic mice, and further investigations are warranted to explore ways for inducing this type of antibody response in the breast milk of HIV-infected women.

Impact of IgA constant domain on HIV-1 neutralizing function of monoclonal antibody F425A1g8.

With the majority of HIV infections resulting from mucosal transmission, induction of an effective mucosal immune response is thought to be pivotal in preventing transmission. HIV-specific IgA, but not IgG, has been detected in the genital tract, seminal fluid, urethral swabs, urine, and vaginal wash samples of HIV-negative sex workers and HIV-status discordant couples. Purified mucosal and plasma IgA from some individuals with highly exposed, persistently seronegative status can neutralize infection and present cross-clade neutralization activity, though present at low levels. We generated a CD4-induced human mAb, F425A1g8, and characterized the impact of its isotype variants on HIV neutralizing activity. The result showed that, in contrast to little neutralization by the F425A1g8 IgG1 in the absence of sCD4, the IgA1 variant of the Ab displayed significant independent neutralization activity against a range of HIV clade B isolates in the absence of sCD4. Studies of the neutralizing function of IgA isotypes, and the functional relationship between different antigenic epitopes and IgA Abs, may also suggest strategies for the intervention of virus transmission and spread within the mucosa of the host, as well as serve to inform the design of vaccine strategies that may be more effective at preventing mucosal transmission. This research clearly suggests that IgA isotype, because of its unique molecular structure, may play an important role in HIV neutralization.

Enhanced neutralization of HIV by antibodies displayed on the S-layer of Caulobacter crescentus.

Innovative methods of prevention are needed to stop the more than two million new HIV-1 infections annually, particularly in women. Local application of anti-HIV antibodies has been shown to be effective at preventing infection in nonhuman primates; however, the concentrations needed are cost prohibitive. Display of antibodies on a particulate platform will likely prolong effectiveness of these anti-HIV agents and lower the cost of goods. Here, we demonstrate that the bacterium Caulobacter crescentus and its highly expressed surface-layer (S-layer) protein can provide this antibody display platform. Caulobacters displaying protein G, alone or with CD4 codisplay, successfully captured HIV-1-specific antibodies and demonstrated functional neutralization. Compared to soluble antibodies, a neutralizing anti-HIV antibody displayed on Caulobacter was as effective or more effective at neutralizing diverse HIV-1 isolates. Moreover, when an antibody reactive with an epitope induced by CD4 binding (CD4i) was codisplayed with CD4, there was significant enhancement in HIV-1 neutralization. These results suggest that caulobacters displaying anti-HIV antibodies offer a distinct improvement in the use of antibodies as microbicides. Furthermore, these reagents can specifically evaluate anti-HIV antibodies in concert with other HIV-1 blocking agents to assess the most suitable tools for conversion to scFvs, allowing for direct display within the S-layer protein and further reducing cost of goods. In summary, C. crescentus, which can be easily produced and chemically stabilized at low cost, is well suited for engineering as an effective platform, offering an inexpensive way to produce and deliver HIV-1-specific microbicides.

A bispecific antibody composed of a nonneutralizing antibody to the gp41 immunodominant region and an anti-CD89 antibody directs broad human immunodeficiency virus destruction by neutrophils.

In addition to the direct neutralization of virus, there is a broader potential for antibody-mediated inhibition of human immunodeficiency virus (HIV) by targeting HIV to effector cells. We demonstrate here that a bispecific antibody incorporating a broadly reactive anti-gp41 antibody, F240, and an anti-IgA receptor (CD89) antibody is effective at directing neutrophils to destroy HIV. Not only are neutrophils the predominant type of white blood cells and very efficient at mediating cell cytotoxicity, they are relatively resistant to infection with HIV. Therefore, they represent a significant weapon against infection if they can be directed and armed to destroy HIV and infected cells.

The neutralization properties of a HIV-specific antibody are markedly altered by glycosylation events outside the antigen-binding domain.

Neutralizing Abs constitute a pivotal mechanism of the adaptive immune response against HIV-1 infection. Yet, most of the Abs that appear in the circulation during HIV infection are nonneutralizing. In this study, we report a dramatic change of the neutralizing properties of a human Ab reactive with the nonneutralizing epitope termed cluster I on the HIV-1 transmembrane protein gp41 when the Ab was produced in Chinese hamster ovary (CHO)-K1 cells. Our laboratory has previously reported that the Ab F240, when produced in a hybridoma, is nonneutralizing as assessed by standard neutralization assays. The F240 IgG1 Ab expressed in CHO cells acquired a strong neutralization activity against a broad range of HIV isolates without a change in immunoreactivity. Sequencing of the F240 mRNAs produced in the parental hybridoma and CHO cells revealed identical sequences, suggesting that acquired neutralization resulted from cell-specific posttranslational modifications. We found that the Ab produced by CHO cells is glycosylated to a greater extent than the parental Ab produced by the hybridoma. Moreover, treatment with peptide N-glycosidase F abrogated F240 neutralization, in an isolate-specific manner, but not Ab b12 neutralization. Interestingly, the F240 isotype-switched variants IgG3 and IgG4, also expressed in CHO cells, exhibited identical immunoreactivity to IgG1 isotypes but had clear differences in viral neutralization. These results suggest that structural features of the Ig molecule other than the primary sequence of the variable regions play a more prominent role in HIV neutralization than anticipated.

Pathogenic human monoclonal antibody against desmoglein 3.

Pemphigus vulgaris (PV) is a potentially fatal autoimmune mucocutaneous disease associated with production of IgG autoantibodies to desmoglein 3 (Dsg3), a 130-kDa epidermal cadherin protein. The binding of pathogenic antibody to Dsg3 on epidermal keratinocytes leads to loss of intercellular adhesion and results in intraepithelial blister formation. Here, we describe a human monoclonal antibody, PVMAB786, a Dsg3-specific IgG4 antibody, from an untreated patient with active PV. The antibody reacts with a 130-kDa protein on keratinocyte cell surfaces and recombinant Dsg3 protein, but not desmoglein 1 protein. PVMAB786 induces acantholysis in normal human skin and mucous membranes and induces a clinical and histological profile similar to human PV when injected into neonatal mice. PVMAB786 will be a valuable tool in identifying the role of Dsg3 in epithelial cell adherence and acantholysis, mechanisms of Dsg3 processing/presentation and V gene and isotype usage in PV pathogenesis.

Human single-chain antibodies inhibit replication of human immunodeficiency virus type 1 (HIV-1).

The F240 human monoclonal antibody specifically recognizes the disulfide loop-bonded immunodominant epitope of gp41 spanning residues 592-606 and expressed broadly on HIV-1 primary isolates. Despite broad reactivity with native virions and HIV-infected cells, the antibody fails to neutralize infection. However, cytoplasmic expression of single-chain antibody (scFv) directed against gp41 of HIV-1 provides a rationale means to inhibit the maturation of envelope protein. The variable regions of the heavy chain and light chain of human monoclonal antibody were amplified by PCR and linked by a 15 amino acid (GGSGS)3 linker in an orientation of VL-linker-VH and retroviral expression vectors were constructed to simultaneously express F240 scFv and eGFP to facilitate selection of scFv-producing cells. Incorporation of a human immunoglobulin signal sequence directed secretion of the F240 scFv (s-scFv) while an otherwise identical vector lacked this sequence (scFv) resulting in intracellular expression of scFv. Transduced human CD4+ H9 T cells were challenged with HIV. While both secreted and nonsecreted F240 scFv inhibited viral production, secretory F240 scFv was more potent. Thus, this novel approach to direct expression of a nonneutralizing scFv using the Ig signal sequence suggests that targeted therapy using antibodies to conserved, highly expressed epitopes may result in a decrease in viral production due to a reduction of viral assembly and/or transport and expression.

Dichotomy in cross-clade reactivity and neutralization by HIV-1 sera: Implications for active and passive immunotherapy.

The identification of broadly reactive and cross-clade neutralizing antibodies will facilitate the development of a more universally effective vaccine for human immunodeficiency virus (HIV). Antibodies in sera from individuals infected with Clade B HIV bind native primary viral isolates, and virus binding correlates with neutralization and stable clinical disease. In this study, we quantified cross-clade antibody reactivity and neutralization by Clades B and C sera. Primary viral isolates were captured by serum IgG bound to anti-human IgG and quantitated as p24 released by lysis of captured virus. Neutralization was determined using PHA-stimulated PBMC. Clade B antibodies reacted more frequently with Clade B R5 virus, but positive sera captured quantitatively more X4 virus than R5 and R5X4 virus. Clade B sera reacted less frequently and captured less Clade C virus than Clade B virus. Antibodies in Clade C sera captured Clades B and C isolates with equal frequency and quantity. There was no difference in neutralization of Clade B virus by either group of sera; however, Clade C sera neutralized Clade C virus, whereas Clade B sera were ineffective against Clade C virus. Thus, there are distinct differences in cross-clade reactivity of and neutralization by antibodies induced in response to Clade C infection compared to Clade B infection. Understanding antibody responses to native virions after Clade C infection and cross clade antibody behavior has implications for understanding pathogenesis and vaccine development.

Binding and neutralization activity of human IgG1 and IgG3 from serum of HIV-infected individuals.

The IgG1 and IgG3 subclasses represent the predominant antibody response to viral infections, including HIV. IgG subclasses differ in their interaction with antigen and functional effects due to specific physiochemical features. With an elongated hinge, IgG3 antibodies tend to have more segmental flexibility, which can render the antibody more effective at interacting with antigen. We have previously shown that the change of the human anti-CD4-binding site monoclonal antibody F105 from IgG1 to IgG3 results in neutralization of a T cell line-adapted isolate (TCLA) resistant to neutralization by the parental IgG1. In the studies presented here, we have purified IgG1 and IgG3 subclasses from the sera of HIV-infected individuals and tested for immunoreactivity with and neutralization of HIV. Purified total IgG3 tended to have less relative reactivity and mediated relatively poorer neutralization of either laboratory or primary isolates. IgG3 also tended to react relatively less well with gp160 and gp120 and more robustly with gp41 and p24. The contrasting results with serum, as opposed to F105, may result from the polyclonal nature of serum antibodies. There is also a failure to make a robust IgG3 response to neutralizing epitopes on envelope glycoproteins during natural infection. These studies suggest that the investigation of isotype effects on neutralization will require isotype-switched human monoclonal antibodies. Understanding isotype and neutralization will provide important data necessary for designing the most effective possible vaccines.

Expression and functional activity of isotype and subclass switched human monoclonal antibody reactive with the base of the V3 loop of HIV-1 gp120.

Immunoglobulins undergo isotype switching in response to antigenic stimulation. The C(H) domains, in particular the hinge region, impose structural constraints on the interaction of antibody with antigen, especially multivalent antigens such as HIV. We previously showed that switching the IgG1 anti-HIV human monoclonal antibody (HMAb) F105 to an IgG3 resulted in significantly enhanced neutralization of HIV. To further investigate the influence of isotype, including the functional activity of HMAbs switched to IgA, which may be important in mucosal defenses, isotype switched antibodies have been generated for the anti-V3 loop base IgG2 HMAb F425B4e8. Reactivity of the IgG1 antibody was greater than the parental IgG2 antibody for SF2 infected cells but less for primary isolate virions. In contrast, there was less reactivity of the IgG3 with either infected cells or virions. IgA reacted significantly more with infected cells and virions as compared to the IgG subclasses. In contrast to previous studies whereby IgG3 enhanced neutralization, comparable neutralization of primary isolate virus was observed for IgG subclasses (IgG1, IgG2, IgG3) and IgA. This may reflect differences in the exposure of epitopes recognized by the HMAb with antibody flexibility being important to neutralization by antibodies reactive with obscured epitopes (e.g., CD4 binding site). Further analysis of the in vitro activity of isotype or subclass switched antibodies, IgA in particular, alone and in combination with other HMAbs, will provide important information on the role of IgG subclass and IgA antibodies on protective immunity to HIV.

Conformational changes in env oligomer induced by an antibody dependent on the V3 loop base.

OBJECTIVE: The HIV-1 env oligomer is structured such that conserved, neutralizing epitopes are obscured by gp120 variable loops. We have studied the ability of an IgG2 human monoclonal antibody (hmAb), F425 B4e8 (B4e8), dependent upon the base of the V3 loop, to induce conformational changes in the env oligomer. DESIGN: The effect of B4e8 antibody on the exposure of neutralizing epitopes and viral neutralization was studied in combination with other hmAb. METHODS: Epitope exposure and viral neutralization was determined using native, intact primary isolate virions. RESULTS: B4e8 antibody neutralizes infection and binds to HIV-infected cells and primary isolate virions. B4e8 and 2G12 enhanced the binding of each other to infected cells or virus and the combination resulted in synergistic neutralization. B4e8 also enhanced the binding of CD4i and CD4 binding site antibodies. CONCLUSIONS: The conserved epitopes exposed by B4e8 are similar to those exposed by the movement of the variable loops following CD4 engagement. Further studies with select antibody combinations should provide important information for the design of effective immunotherapeutic agents.

Interactions of human antibodies, epitope exposure, antibody binding and neutralization of primary isolate HIV-1 virions.

OBJECTIVE: Development of an effective HIV vaccine has been limited because of the inherent structural properties of the HIV envelope on native virions and the failure of the immune system to respond in an effective manner. Identification of the interactions of human antibodies with virions resulting in neutralization will facilitate vaccine design. DESIGN: Combinations of human monoclonal antibodies (hMAb) were studied for binding to and neutralization of primary isolate virions. METHODS: Virion binding and neutralization were measured using primary isolate virions. RESULTS: Antibodies and combinations of antibodies to epitopes exposed upon CD4 binding (CD4i) and V3 loop antibodies resulted in additive binding and neutralization of R5X4 virus. Antibodies did not bind to or neutralize R5 virus as well. The combination of V3 loop antibody with 2G12 resulted in enhanced neutralization and binding to the R5X4 isolate but not the R5 isolate. Preincubation of the R5X4 isolate with F240, a non-neutralizing anti-gp41 antibody, significantly enhanced binding and neutralization by CD4i hMAb and 2F5. F240 also enhanced the binding of 2F5 to the R5 isolate and the neutralization of the R5 isolate mediated by 2G12. CONCLUSIONS: Neutralizing epitopes are obscured on intact primary isolate virions and are dynamically exposed upon ligand (CD4) interactions. Interestingly, a non-neutralizing antibody to gp41 also increased binding and neutralizing activity of some hMAb that poorly neutralized R5 virus. These data suggest that non-neutralizing epitopes may be appropriate targets for vaccine design and epitope exposure should be considered in the development of immunotherapeutic strategies for HIV.

Evidence of determinant spreading in the antibody responses to prostate cell surface antigens in patients immunized with prostate-specific antigen.

PURPOSE: Prostate cancer consistently remains a difficult clinical problem. The development of novel therapy strategies for effective control and treatment of prostate cancer is essential. The prostate represents a unique site for immunotherapy, in part because prostate-specific immunity would most probably be without significant long-term sequellae. Antibodies and cell-mediated immunity, induced by either active or passive immunization, represent potential means to specifically target prostate tumor cells. EXPERIMENTAL DESIGN: The serum IgG response to cell surface antigens expressed on LNCAP [prostate-specific antigen (PSA)-positive] and PC-3 (PSA-negative) were analyzed in individuals with advanced disease receiving vaccinia- or fowlpox-expressed PSA (v-PSA or f-PSA, respectively) by flow cytometry. RESULTS: Sera from all seven patients in a Phase I study of v-PSA, collected prior to the third immunization, reacted with both prostate tumor cell lines. The majority of individuals (n = 12) in a Phase II trial of v-PSA and f-PSA developed sustainable antibody responses to cell surface antigens on the prostate tumor cell lines. The magnitude and kinetics of these responses were dependent on the immunization schedule. Of importance, the baseline serum of only one of nine patients tested had reactivity with nonprostate tumor cell lines. Sera from three normal males also lacked reactivity with prostate tumor cells. CONCLUSIONS: PSA vaccine constructs are immunogenic and induce antibody responses to a multitude of surface antigens on prostate tumor cell lines by epitope or determinant spreading after stimulation of the immune system by PSA immunization.