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Lyme disease is a tick-borne, multisystem infection caused by the spirochete Borreliella burgdorferi. Although Abs have been implicated in the resolution of Lyme disease, the specific B cell epitopes targeted during human infections remain largely unknown. In this study, we characterized and defined the structural epitope of a patient-derived bactericidal monoclonal IgG (B11) against outer surface protein C (OspC), a homodimeric lipoprotein necessary for B. burgdorferi tick-mediated transmission and early-stage colonization of vertebrate hosts. High-resolution epitope mapping was accomplished through hydrogen deuterium exchange-mass spectrometry and X-ray crystallography. Structural analysis of B11 Fab-OspCA complexes revealed the B11 Fabs associated in a 1:1 stoichiometry with the lateral faces of OspCA homodimers such that the Abs are essentially positioned perpendicular to the spirochete's outer surface. B11's primary contacts reside within the membrane-proximal regions of α-helices 1 and 6 and adjacent loops 5 and 6 in one OspCA monomer. In addition, B11 spans the OspCA dimer interface, engaging opposing α-helix 1', α-helix 2', and loop 2-3' in the second OspCA monomer. The B11-OspCA structure is reminiscent of the recently solved mouse transmission blocking monoclonal IgG B5 in complex with OspCA, indicating a mode of engagement with OspC that is conserved across species. In conclusion, we provide a detailed insight into the interaction between a functional human Ab and an immunodominant Lyme disease Ag long considered an important vaccine candidate., (Copyright © 2024 by The American Association of Immunologists, Inc.)

COVID-19 disproportionately affected minorities, while research barriers to engage underserved communities persist. Serological studies reveal infection and vaccination histories within these communities, however lack of consensus on downstream evaluation methods impede meta-analyses and dampen the broader public health impact. To reveal the impact of COVID-19 and vaccine uptake among diverse communities and to develop rigorous serological downstream evaluation methods, we engaged racial and ethnic minorities in Massachusetts in a cross-sectional study (April-July 2022), screened blood and saliva for SARS-CoV-2 and human endemic coronavirus (hCoV) antibodies by bead-based multiplex assay and point-of-care (POC) test and developed across-plate normalization and classification boundary methods for optimal qualitative serological assessments. Among 290 participants, 91.4% reported receiving at least one dose of a COVID-19 vaccine, while 41.7% reported past SARS-CoV-2 infections, which was confirmed by POC- and multiplex-based saliva and blood IgG seroprevalences. We found significant differences in antigen-specific IgA and IgG antibody outcomes and indication of cross-reactivity with hCoV OC43. Finally, 26.5% of participants reported lingering COVID-19 symptoms, mostly middle-aged Latinas. Hence, prolonged COVID-19 symptoms were common among our underserved population and require public health attention, despite high COVID-19 vaccine uptake. Saliva served as a less-invasive sample-type for IgG-based serosurveys and hCoV cross-reactivity needed to be evaluated for reliable SARS-CoV-2 serosurvey results. The use of the developed rigorous downstream qualitative serological assessment methods will help standardize serosurvey outcomes and meta-analyses for future serosurveys beyond SARS-CoV-2., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Binder et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

319-44 is a human monoclonal antibody capable of passively protecting mice against tick-mediated infection with Borreliella burgdorferi, the bacterial genospecies responsible for Lyme disease in North America. In vitro, 319-44 has complement-dependent borreliacidal activity and spirochete agglutinating properties. Here, we report the 2.2 Å-resolution crystal structure of 319-44 Fab fragments in complex with Outer surface protein A (OspA), the ~30 kDa lipoprotein that was the basis of the first-generation Lyme disease vaccine approved in the United States. The 319-44 epitope is focused on OspA β-strands 19, 20, and 21, and the loops between β-strands 16-17, 18-19, and 20-21. Contact with loop 20-21 explains competition with LA-2, the murine monoclonal antibody used to estimate serum borreliacidal activities in the first-generation Lyme disease vaccine clinical trials. A high-resolution B-cell epitope map of OspA will accelerate structure-based design of second generation OspA-based vaccines., (© 2023 Wiley Periodicals LLC.)

Enterotoxigenic Escherichia coli (ETEC) is a common cause for diarrheal infections in children in low- and middle-income countries (LMICs). To date, no ETEC vaccine candidates have been approved. Passive immunization with low-cost, oral formulations of secretory IgA (sIgA) against ETEC is an alternative approach to protect high-risk populations in LMICs. Using a model sIgA monoclonal antibody (anti-LT sIgA2-mAb), the stability profiles of different formulations were assessed during storage and in in vitro digestion models (mimicking in vivo oral delivery). First, by employing various physicochemical techniques and a LT-antigen binding assay, three formulations with varying acid-neutralizing capacity (ANC) were evaluated to stabilize sIgA2-mAb during stress studies (freeze-thaw, agitation, elevated temperature) and during exposure to gastric phase digestion. Next, a low-volume, in vitro intestinal digestion model was developed to screen various additives to stabilize sIgA2-mAb in the intestinal phase. Finally, combinations of high ANC buffers and decoy proteins were assessed to collectively protect sIgA2-mAb during in vitro sequential (stomach to intestine) digestion. Based on the results, we demonstrate the feasibility of low-cost, 'single-vial', liquid formulations of sIgA-mAbs delivered orally after infant feeding for passive immunization, and we suggest future work based on a combination of in vitro and in vivo stability considerations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The Lyme disease (LD) vaccine formerly approved for use in the United States consisted of recombinant outer surface protein A (OspA) from Borrelia burgdorferi sensu stricto (ss), the bacterial genospecies responsible for the vast majority of LD in North America. OspA is an ∼30 kDa lipoprotein made up of 21 antiparallel β-strands and a C-terminal α-helix. In clinical trials, protection against LD following vaccination correlated with serum antibody titers against a single epitope near the C-terminus of OspA, as defined by the mouse monoclonal antibody (MAb), LA-2. However, the breadth of the human antibody response to OspA following vaccination remains undefined even as next-generation multivalent OspA-based vaccines are under development. In this report, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes recognized by a unique panel of OspA human MAbs, including four shown to passively protect mice against experimental B. burgdorferi infection and one isolated from a patient with antibiotic refractory Lyme arthritis. The epitopes grouped into three spatially distinct bins that, together, encompass more than half the surface-exposed area of OspA. The bins corresponded to OspA β-strands 8-10 (bin 1), 11-13 (bin 2), and 16-20 plus the C-terminal α-helix (bin 3). Bin 3 was further divided into sub-bins relative to LA-2's epitope. MAbs with complement-dependent borreliacidal activity, as well as B. burgdorferi transmission-blocking activity in the mouse model were found within each bin. Therefore, the resulting B cell epitope map encompasses functionally important targets on OspA that likely contribute to immunity to B. burgdorferi .

Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Humphries, Girardin, Wallace, Ejemel, Amcheslavsky, McMahon, Schiller, Ma, Cruz, Dupuis, Payne, Maryam, Yilmaz, McDonough, Pierce, Schiffer, Kruse, Klempner, Cavacini, Fitzgerald and Wang.)

The endocervix, the primary site of Chlamydia trachomatis (Ct) infection in women, has a unique repertoire of locally synthesized IgG and secretory IgA (SIgA) with contributions from serum IgG. Here, we assessed the ability of genital and serum-derived IgG and IgA from women with a recent positive Ct test to neutralize Ct elementary bodies (EBs) and inhibit inclusion formation in vitro in human endocervical epithelial cells. We also determined if neutralization was influenced by the major outer membrane protein (MOMP) of the infecting strain, as indicated by ompA gene sequencing and genotyping. At equivalent low concentrations of Ct EB (D/UW-3/Cx + E/UW-5/Cx)-specific antibody, genital-derived IgG and IgA and serum IgA, but not serum IgG, significantly inhibited inclusion formation, with genital IgA being most effective, followed by genital IgG, then serum IgA. The well-characterized Ct genotype D strain, D/UW-3/Cx, was neutralized by serum-derived IgG from patients infected with genotype D strains, genital IgG from patients infected with genotype D or E strains, and by genital IgA from patients infected with genotype D, E, or F strains. Additionally, inhibition of D/UW-3/Cx infection by whole serum, rather than purified immunoglobulin, was associated with levels of serum EB-specific IgG rather than the genotype of infecting strain. In contrast, a Ct genotype Ia clinical isolate, Ia/LSU-56/Cx, was neutralized by whole serum in a genotype and genogroup-specific manner, and inhibition also correlated with EB-specific IgG concentrations in serum. Taken together, these data suggest that (i) genital IgA most effectively inhibits Ct infection in vitro, (ii) human antibody-mediated inhibition of Ct infection is significantly influenced by the ompA genotype of the infecting strain, (iii) the genital antibody repertoire develops or matures differently compared to systemic antibody, and (iv) ompA genotype-specificity of inhibition of infection by whole serum can be overcome by high concentrations of Ct-specific IgG., Competing Interests: The authors have declared that no competing interests exist.

Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.

Immunoglobulin A (IgA) antibodies are critical to mucosal protection, specifically dimeric IgA (dIgA) and secretory IgA (sIgA), which rely on the J chain to polymerize. There is an absence of monoclonal antibodies that can specifically bind to polymeric IgA without the need to denature the molecule. We generated a panel of highly specific mouse anti-J chain antibodies that react with both intact and denatured nonhuman primate dIgA and human dIgA and sIgA of both the IgA1 and IgA2 subclass. We expanded use of this antibody for quantification of dIgA and sIgA using biolayer interferometry or enzyme-linked immunosorbent assay and use for affinity chromatography. This is a significant improvement over available anti-IgA antibodies in the field, which will allow for expanded use in clinical testing.

Mucosal surfaces of the gastrointestinal tract play an important role in immune homeostasis and defense and may be compromised by enteric disorders or infection. Therapeutic intervention using monoclonal antibody (mAb) offers the potential for treatment with minimal off-target effects as well as the possibility of limited systemic exposure when administered orally. Critically, to achieve efficacy at luminal surfaces, mAb must remain stable and functionally active in the gastrointestinal environment. To better understand the impact of isotype, class, and molecular structure on the intestinal stability of recombinant antibodies, we used an in vitro simulated intestinal fluid (SIF) assay to evaluate a panel of antibody candidates for enteric mAb-based therapeutics. Recombinant IgG1 was the least stable following SIF incubation, while the stability of IgA generally increased upon polymerization, with subtle differences between subclasses. Notably, patterns of variability within and between mAbs suggest that variable regions contribute to mAb stability and potentially mediate mAb susceptibility to proteases. Despite relatively rapid degradation in SIF, mAbs targeting Enterotoxigenic Escherichia coli (ETEC) displayed functional activity following SIF treatment, with SIgA1 showing improved function compared to SIgA2. The results of this study have implications for the design of enteric therapeutics and subsequent selection of lead candidates based upon in vitro intestinal stability assessments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea-associated illness in developing countries. There is currently no vaccine licensed to prevent ETEC and the development of an efficacious prophylaxis would provide an intervention with significant impact. Recent studies suggested that effective protection could be achieved by inducing immunity to block colonization of ETEC. Here, we evaluated the efficacy of secretory (s) IgA2 and dimeric (d) IgA2 of an anti-colonization factor antigen antibody, 68-61, in the Aotus nancymaae nonhuman primate (NHP) ETEC challenge model via oral and parental delivery. Thirty-nine animals were distributed across 3 groups of 13, and challenged with 5.0x10 11 colony forming unit (CFU) of H10407 on Day 0. Group 1 received a dIgA2 68-61 subcutaneously on day 0. Group 2 received a SIgA2 68-61 orally on days -1, 0, and +1, and Group 3 received an irrelevant SIgA2 antibody orally on days -1, 0, and +1. All animals were observed for symptoms of diarrhea, and stools were collected for ETEC colony counts. Anti-CfaE SIgA2 treatment significantly lowered the attack rate, resulting in a protective efficacy of 74.1% (p = 0.025) in Group 2 as compared to Group 3. The anti-CfaE dIgA2 treatment group had reduced diarrheal attack rate, although the reduction did not reach significance (57.1%; p = 0.072) as compared to the irrelevant SIgA2 Group 3. Our results demonstrated the feasibility of oral administration of SIgA as a potential immunoprophylaxis against enteric infections. To our knowledge, this is the first study to demonstrate the efficacy of administrated SIgA in a nonhuman primate model., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 IL2rγ null 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., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Enterotoxigenic Escherichia coli (ETEC) causes diarrheal illness in infants in the developing world and travelers to countries where the disease is endemic, including military personnel. ETEC infection of the host involves colonization of the small intestinal epithelium and toxin secretion, leading to watery diarrhea. There is currently no vaccine licensed to prevent ETEC infection. CFA/I is one of the most common colonization factor antigens (CFAs). The CFA/I adhesin subunit, CfaE, is required for ETEC adhesion to host intestinal cells. Human antibodies against CfaE have the potential to block colonization of ETEC and serve as an immunoprophylactic against ETEC-related diarrhea. Mice transgenic for human immunoglobulin genes were immunized with CfaE to generate a panel of human monoclonal IgG1 antibodies (HuMAbs). The most potent IgG1 antibodies identified in the in vitro functional assays were selected and isotype switched to secretory IgA (sIgA) and tested in animal colonization assays via oral administration. Over 300 unique anti-CfaE IgG1 HuMAbs were identified. The lead IgG1 anti-CfaE HuMAbs completely inhibited hemagglutination and blocked adhesion of ETEC to Caco-2 cells. Epitope mapping studies revealed that HuMAbs recognized epitopes in the N-terminal domain of CfaE near the putative receptor binding site. Oral administration of anti-CfaE antibodies in either IgG or sIgA isotypes inhibited intestinal colonization in mice challenged with ETEC. A 2- to 4-log decrease in CFU was observed in comparison to mice challenged with irrelevant isotype controls. We identified fully human monoclonal antibodies against the CfaE adhesion domain that can be potentially employed as an immunoprophylactic to prevent ETEC-related diarrhea., (Copyright © 2018 Giuntini et al.)

Pemphigus vulgaris (PV) is characterized by IgG1 and IgG4 autoantibodies to desmoglein (Dsg) 3, causing suprabasal blistering of skin and mucous membranes. IgG4 is the dominant autoantibody subclass in PV and correlates with disease activity, whereas IgG1 can be associated with remittent disease. It is unknown if switching the same variable region between IgG4 and IgG1 directly impacts pathogenicity. Here, we tested whether three pathogenic PV monoclonal antibodies (mAbs) from three different patients demonstrate differences in antigen affinity, epitope specificity, or pathogenicity when expressed as IgG1 or IgG4. F706 anti-Dsg3 IgG4 and F779 anti-Dsg3 IgG1, previously isolated as heterohybridomas, and Px43, a monovalent anti-Dsg3/Dsg1 IgG antibody isolated by phage display, were subcloned to obtain paired sets of IgG1 and IgG4 mAbs. Using ELISA and cell surface staining assays, F706 and F779 demonstrated similar antigen binding affinities of IgG1 and IgG4, whereas Px43 showed 3- to 8-fold higher affinity of IgG4 versus IgG1 by ELISA, but identical binding affinities to human skin, perhaps due to targeting of a quaternary epitope best displayed in tissues. All 3 mAb pairs targeted the same extracellular cadherin (EC) domain on Dsg3, caused Dsg3 internalization in primary human keratinocytes, and caused suprabasal blisters in human skin at comparable doses. We conclude that switching IgG1 and IgG4 subclasses of pathogenic PV mAbs does not directly affect their antigen binding or pathogenic properties.

Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies., (Copyright © 2016, American Association for the Advancement of Science.)

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.

Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies (autoAbs) against desmoglein 3 (Dsg3). Here, we clone anti-Dsg3 antibodies (Abs) from four PV patients and identify pathogenic VH1-46 autoAbs from all four patients. Unexpectedly, VH1-46 autoAbs had relatively few replacement mutations. We reverted antibody somatic mutations to their germline sequences to determine the requirement of mutations for autoreactivity. Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs. Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding. Our data suggest that VH1-46 autoantibody gene usage is commonly found in PV because VH1-46 Abs require few to no mutations to acquire Dsg3 autoreactivity, which may favour their early selection. Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

The HIV/AIDS pandemic remains an enormous global health concern. Despite effective prevention options, 2.6 million new infections occur annually, with women in developing countries accounting for more than half of these infections. New prevention strategies that can be used by women are urgently needed. Topical microbicides specific for HIV-1 represent a promising prevention strategy. Conceptually, using harmless bacteria to display peptides or proteins capable of blocking entry provides an inexpensive approach to microbicide development. To avoid the potential pitfalls of engineering commensal bacteria, our strategy is to genetically display infection inhibitors on a non-native bacterium and rely on topical application of stabilized bacteria before potential virus exposure. Due to the high density cell-surface display capabilities and the inherent low toxicity of the bacterium, the S-layer mediated protein display capabilities of the non-pathogenic bacterium Caulobacter crescentus has been exploited for this approach. We have demonstrated that C. crescentus displaying MIP1α or CD4 interfered with the virus entry pathway and provided significant protection from HIV-1 pseudovirus representing clade B in a standard single cycle infection assay. Here we have expanded our C. crescentus based microbicide approach with additional and diverse classes of natural and synthetic inhibitors of the HIV-1 entry pathway. All display constructs provided variable but significant protection from HIV-1 infection; some with protection as high as 70%. Further, we describe protection from infection with additional viral clades. These findings indicate the significant potential for engineering C. crescentus to be an effective and readily adaptable HIV-1 microbicide platform.

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.

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.

The development of alternative strategies to prevent HIV infection is a global public health priority. Initial efforts in anti-HIV microbicide development have met with poor success as the strategies have relied on a non-specific mechanism of action. Here, we report the development of a microbicide aimed at specifically blocking HIV entry by displaying molecular components of the HIV/host cell attachment complex on the surface of Caulobacter crescentus, a harmless aquatic bacterium. This bacterium can be readily manipulated to present heterologous proteins at high density on its surface by genetic insertion into its crystalline surface layer protein. In separate constructions, we generated bacteria displaying domain 1 of CD4 and MIP1alpha. Each moiety reacted with specific antibodies by Western immunoblot and immuno-fluorescence microscopy. Microbicide functionality was assessed using an HIV pseudotype virus assay system representing Clade B subtypes. Bacteria displaying MIP1alpha reduced infectivity by 35-78% depending on the specific subtype while CD4 display reduced infection by as much as 56%. Combinations of both constructs reduced infectivity by nearly 98%. We demonstrated that HIV infection could be inhibited using a strategy aimed at HIV-specific molecular interactions with Caulobacter surface protein display, and that sufficient protein folding and conformation could be mimicked to bind and block entry. Further, this is the first demonstration that Caulobacter surface protein display may be a useful approach to preventing HIV infection or other viruses as a microbicide. We propose that this harmless bacterium, which is inexpensive to produce and formulate, might be suitable for topical applications as a viable alternative in the search for effective microbicides to counteract the world wide incidence of HIV infection.

The V3 loop of HIV-1 gp120 is considered occluded on many primary viruses. However, virus sensitivity to neutralization by different V3 mAbs often varies, indicating that access to V3 is not restricted equally for all antibodies. Here, we have sought to gain a better understanding of these restrictions by determining the neutralizing activities of 7 V3 mAbs (19b, 39F, CO11, F2A3, F530, LA21, and LE311) against 15 subtype B primary isolates and relating these activities to the fine specificity of the mAbs. Not surprisingly, we found that most mAbs neutralized the same 2-3 viruses, with only mAb F530 able to neutralize 2 additional viruses not neutralized by the other mAbs. Epitope mapping revealed that positively-charged residues in or near the V3 stem are important for the binding of all the mAbs and that most mAbs seem to require the Pro residue that forms the GPGR beta hairpin turn in the V3 tip for binding. Based on the mapping, we determined that V3 sequence variation accounted for neutralization resistance of approximately half the viruses tested. Comparison of these results to those of select V3 mAbs with overall better neutralizing activities in the light of structural information illustrates how an antibody's mode of interaction with V3, driven by contact residue requirements, may restrict the antibody from accessing its epitope on different viruses. Based on the data we propose an angle of interaction with V3 that is less stringent on access for antibodies with cross-neutralizing activity compared to antibodies that neutralize relatively fewer viruses.

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.

F425-B4e8 (B4e8) is a monoclonal antibody isolated from a human immunodeficiency virus type 1 (HIV-1)-infected individual that recognizes the V3 variable loop on the gp120 subunit of the viral envelope spike. B4e8 neutralizes a subset of HIV-1 primary isolates from subtypes B, C and D, which places this antibody among the very few human anti-V3 antibodies with notable cross-neutralizing activity. Here, the crystal structure of the B4e8 Fab' fragment in complex with a 24-mer V3 peptide (RP142) at 2.8 A resolution is described. The complex structure reveals that the antibody recognizes a novel V3 loop conformation, featuring a five-residue alpha-turn around the conserved GPGRA apex of the beta-hairpin loop. In agreement with previous mutagenesis analyses, the Fab' interacts primarily with V3 through side-chain contacts with just two residues, Ile(P309) and Arg(P315), while the remaining contacts are to the main chain. The structure helps explain how B4e8 can tolerate a certain degree of sequence variation within V3 and, hence, is able to neutralize an appreciable number of different HIV-1 isolates.

The identification of cross-neutralizing antibodies to HIV-1 is important for designing antigens aimed at eliciting similar antibodies upon immunization. The monoclonal antibody (mAb) F425-B4e8 had been suggested previously to bind an epitope at the base of V3 and shown to neutralize two primary HIV isolates. Here, we have assessed the neutralization breadth of mAb F425-B4e8 using a 40-member panel of primary HIV-1 and determined the epitope specificity of the mAb. The antibody was able to neutralize 8 clade B viruses (n=16), 1 clade C virus (n=11), and 2 clade D viruses (n=6), thus placing it among the more broadly neutralizing anti-V3 antibodies described so far. Contrary to an initial report, results from our scanning mutagenesis of the V3 region suggest that mAb F425-B4e8 interacts primarily with the crown/tip of V3, notably Ile(309), Arg(315), and Phe(317). Despite the somewhat limited neutralization breadth of mAb F425-B4e8, the results presented here, along with analyses from other cross-neutralizing anti-V3 mAbs, may facilitate the template-based design of antigens that target V3 and permit neutralization of HIV-1 strains in which the V3 region is accessible to antibodies.

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.

In a primate model of postnatal virus transmission, we have previously shown that 1 h post-exposure prophylaxis (PEP) with a triple combination of neutralizing monoclonal antibodies (nmAbs) conferred sterilizing protection to neonatal macaques against oral challenge with pathogenic simian-human immunodeficiency virus (SHIV). Here, we show that nmAbs can also partially protect SHIV-exposed newborn macaques against infection or disease, when given as 12 or 24 h PEP, respectively. This work delineates the potential and the limits of passive immunoprophylaxis with nmAbs. Even though 24 h PEP with nmAbs did not provide sterilizing immunity to neonatal monkeys, it contained viremia and protected infants from acute disease. Taken together with our results from other PEP studies, these data show that the success of passive immunization depends on the nmAb potency/dose and the time window between virus exposure and start of immunotherapy.

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.

Carbohydrate antigens are important targets of the immune system in clearing bacterial pathogens. Although the immune system almost exclusively uses antibodies in response to foreign carbohydrates, there is still much to learn about the role of different epitopes on the carbohydrate as targets of protective immunity. We examined the role of acetyl group-dependent and -independent epitopes on the staphylococcal surface of polysaccharide poly-N-acetylated glucosamine (PNAG) by use of human monoclonal antibodies (MAbs) specific for such epitopes. We utilized hybridoma technology to produce fully human immunoglobulin G2 (IgG2) MAbs from B cells of an individual post-Staphylococcus aureus infection and cloned the antibody variable regions to produce an IgG1 form of each original MAb. Specificity and functionality of the purified MAbs were tested in vitro using enzyme-linked immunosorbent assays, complement deposition, and opsonophagocytic assays. We found that a MAb (MAb F598) that bound the best to nonacetylated or backbone epitopes on PNAG had superior complement deposition and opsonophagocytic activity compared to two MAbs that bound optimally to PNAG that was expressed with a native level (>90%) of N-acetyl groups (MAbs F628 and F630). Protection of mice against lethality due to S. aureus strains Mn8 and Reynolds further showed that the backbone-specific MAb had optimal protective efficacy compared with the acetate-specific MAbs. These results provide evidence for the importance of epitope specificity in inducing the optimal protective antibody response to PNAG and indicate that MAbs to the deacetylated form of PNAG could be immunotherapeutic agents for preventing or treating staphylococcal infections.

We have determined the crystal structure of the Fab fragment from F105, a broadly reactive human antibody with limited potency that recognizes the CD4 binding site of gp120. The structure reveals an extended CDR H3 loop with a phenylalanine residue at the apex and shows a striking pattern of serine and tyrosine residues. Modeling the interaction between gp120 and F105 suggests that the phenylalanine may recognize the binding pocket of gp120 used by Phe(43) of CD4 and that numerous tyrosine and serine residues form hydrogen bonds with the main chain atoms of gp120. A comparison of the F105 structure to that of immunoglobulin G1 b12, a much more potent and broadly neutralizing antibody with an overlapping epitope, suggests similarities that contribute to the broad recognition of human immunodeficiency virus by both antibodies. While the putative epitope for F105 shows significant overlap with that predicted for b12, it appears to differ from the b12 epitope in extending across the interface between the inner and outer domains of gp120. In contrast, the CDR loops of b12 appear to interact predominantly with the outer domain of gp120. The difference between the predicted epitopes for b12 and F105 suggests that the unique potency of b12 may arise from its ability to avoid the interface between the inner and outer domains of gp120.

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., (Copyright 2005 Wiley-Liss, Inc.)

CD40 is expressed on basal keratinocytes and Squamous Cell Cancer of the Head and Neck (SCCHN) tumor cells in vivo and in vitro. CD40 ligation reduces proliferation of SCCHN cell lines and enhances EGFr mediated inhibition of proliferation. We investigated the mechanisms of CD40 function and EGFr cross-communication in SCCHN cell lines. CD40 ligation inhibited spontaneous and Fas-induced apoptosis. CD40 ligation specifically increased the secretion of IL-8, VEGF and PGE(2) but not IL-6, IL-10, FasL, GM-CSF, or TGFalpha. Co-ligation with EGFr further increased IL-8, VEGF and PGE(2) secretion. CD40 ligation also induced delayed activation and tyrosine phosphorylation of EGFr. CD40 induces secretion of specific proinflammatory and proangiogenic cytokines, inhibits spontaneous and Fas-induced apoptosis and increases EGFr phosphorylation. CD40 signaling may enhance the survival of SCCHN and tumor stroma.

Squamous cell cancer of the head and neck (SCCHN) is associated with production of pro-inflammatory and pro-angiogenic cytokines. We hypothesized that cytokine serum levels will correlate with tumor volume and aggressiveness. We investigated interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and epidermal growth factor receptor (EGFR) in SCCHN. The patient population consisted of normal and irradiated controls: patients with newly diagnosed SCCHN, and patients with recurrent or metastatic disease. Pretreatment sera were studied by ELISA. Serum IL-8 levels, as opposed to VEGF or EGFR, were consistently elevated in patients with recurrent or metastatic disease. The differences in mean serum IL-8, compared to controls, were significant (p=0.02). Serum levels of IL-8 are consistently elevated in patients with recurrent or metastatic SCCHN and elevated levels may correlate with advanced or aggressive disease. Further, more intensive, study of IL-8 as a biomarker in SCCHN is warranted.

Human immunodeficiency virus type 1 (HIV-1) is phylogenetically classified into groups and clades (or subtypes). Human neutralizing monoclonal antibodies (nMAbs), originally isolated from individuals infected with HIV-1 group M-clade B, neutralized not only primary HIV-1 clade B isolates in vitro but also primary isolates of other group M clades (A, C, D, E, and F). This corrected the previously held notion that primary HIV-1 isolates are resistant to neutralizing antibodies. Here we show that anti-HIV-1 group M-clade B nMAbs potently neutralized primary isolates of the phylogenetically distant HIV-1 group O. We and others have previously shown that passive immunization with human nMAbs protected adult or neonatal primates against infection with simian-human immunodeficiency virus strains encoding HIV-1 group M-clade B envelope genes. The in vitro cross-group neutralization shown here underscores the broad potential of these nMAbs against divergent virus variants and the relevance of their epitopes in the design of acquired immunodeficiency syndrome vaccines.

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.

Background: The majority of infants infected through maternal transmission acquire the virus during birth or postpartum through breastfeeding: mucosal exposure is considered to be a major route of infection., Objectives: To develop passive immunization with human neutralizing monoclonal antibodies (mAbs) against mother-to-child transmission of HIV during delivery and through breastfeeding., Design: An oral challenge model in newborn rhesus macaques mimicked peri- and postpartum virus transmission., Methods: Neonatal rhesus macaques were challenged orally with the highly pathogenic, chimeric simian-human immunodeficiency virus SHIV89.6P and given post-exposure prophylaxis with a quadruple combination of neutralizing human mAbs, IgG1b12, 2G12, 2F5, and 4E10, directed against conserved epitopes of HIV envelope glycoproteins. Control animals were virus challenged but left untreated. All infants were followed prospectively for signs of viremia and immunodeficiency., Results: Two out of four macaque infants treated with neutralizing mAbs showed no evidence of infection; the other two maintained normal CD4 T cell counts. In contrast, all control animals became highly viremic and had profound CD4 T cell losses; three out of four died from AIDS within 1.5-6 weeks of the challenge., Conclusions: Passive immunization with this quadruple neutralizing mAbs combination may represent a promising approach to prevent peri- and postnatal HIV transmission. Furthermore, the epitopes recognized by the four neutralizing mAbs are key determinants to achieve complete protection and represent important targets against which to develop active, antibody-response-based AIDS vaccines.

We investigated the ability of several human neutralizing monoclonal antibodies (nmAbs), originally raised against human immunodeficiency virus (HIV) clade B isolates, to neutralize primary clade A and D isolates as single agents and in combinations. All four primary HIV clade A isolates and five primary HIV clade D isolates tested were neutralized >99% by the quadruple combination of nmAbs IgG1b12, 2G12, 2F5, and 4E10. These mAbs recognize conserved epitopes on HIV-1 envelope (Env), resulting in strong cross-clade neutralization. Previously, we showed synergistic neutralization of primary HIV-1 clade C isolates in vitro by the same nMAb combination. We and others also showed neutralization of primary HIV clade B strains. Together, our data show that the quadruple combination of mAbs effectively neutralized primary HIV clade A, B, C, and D isolates.

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.

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.

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.

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.

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.