Change in Anti- gp120 Human Monoclonal Antibody Isotype Significantly Improves HIV-1 Neutralization

HIV vaccine efforts tend to focus on the induction of IgG neutralizing antibodies. In part, this may stem from the observations that most HIV infected individuals fail to produce significant mucosal IgA. However, this is unlike most other infections and in turn it can be argued that mucosal IgA with appropriate function and specificity may contribute significantly to the prevention of HIV transmission. To explore this, we previously isotype switched F425A1g8, an anti-HIV CD4i human monoclonal antibody that binds to epitopes exposed upon CD4 binding (CD4i) The VH and VL chains were amplified from the IgG hybridoma and inserted into IgA1 or IgA2 and IgKappa vectors respectively. Stable cells lines were produced and antibody was collected and purified. Initial results showed that the IgA1 variant neutralized a number of HIV-1 isolates better than its parental form IgG1. We believe the increased neutralization of HIV is mainly due to the structural differences between IgG1 and IgA1. We hypothesize that the extended hinge of IgA1 may increase segmental flexibility and change the interaction of antibody with CD4i epitopes of the HIV, resulting in greater avidity. To look at this further, we have generated monomeric and dimeric IgA1 and IgA2 variants of three different CD4i antibodies: F425A1g8, 17b and E51. All antibody variants will be tested for immumoreactivity, HIV neutralization, prevention of transmission and ADCVI activity. Consistent with our previous results, there are significant differences in functional activity of the other CD4i antibodies with IgA1 more effective than the IgA2 variants. Additional activities will contribute to the hypothesis that the extended hinge region of the IgA1 antibody increases the antibodies ability to access the CD4i epitopes upon HIV-1 binding to CD4. These studies should impact on the design of active and passive immunotherapy and the prevention of HIV transmission

Human monoclonal antibodies to Plasmodium falciparum circumsporozoite protein for transient passive protection of malaria travelers to endemic areas

Plasmodium falciparum, is a protozoa that causes over 214 million cases of Malaria worldwide and the World Health Organization reported an estimated 438,000 deaths attributed to malaria in 2015. Current prevention strategies have reduced malaria cases but they are either costly, have poor efficacy or resistance has begun to develop. There is a global need for an effective pre-exposure prophylaxis treatment. The leading Malaria vaccine candidate is RTS,S which contains a monovalent Plasmodium falciparum circumsporozoite protein (CSP). The goal of this vaccine is to induce anti-CSP antibodies that would block sporozoite invasion of hepatocytes and thereby hinder parasite development into a blood-stage infection that causes malaria morbidity and mortality. Antibodies isolated from individuals who have received the RTS,S vaccine have been shown to prevent infection of hepatocytes, suggesting that CSP antibodies could be used prophylactically. However, phase III trial results of the vaccine have shown underwhelming efficacy in children. Growing resistance to transient protection strategies for travelers and low efficacy in vaccine trials suggest there is a need for a new treatment strategy. The generation of CSP specific human monoclonal antibodies (mAbs) would be useful as prevention especially for individuals that are temporarily exposed to Malaria in endemic regions such as travelers or military personnel. Isolation and production of therapeutic mAbs traditionally utilizes a handful of techniques including antibody engineering, phage display or hybridoma generation from transgenic mice. We have sorted antigen-specific memory B-cells from the peripheral blood of children naturally infected with malaria to isolate CSP-specific memory B-cells. These cells were individually sorted and PCR was performed to amplify antibody variable regions of the B-cell’s antibody mRNA. Samples that produced heavy and light chain antibody sequence were cloned and transiently expressed. We plan to characterize these mAbs for binding and neutralization of CSP to identify functional therapeutic mAbs

Development of an HIV-1 Specific Microbicide Using Caulobacter crescentus S-Layer Mediated Display of CD4 and MIP1α

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 [1], [2]. In separate constructions, we generated bacteria displaying domain 1 of CD4 and MIP1α. 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 MIP1α 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

IgA as a potential candidate for enteric monoclonal antibody therapeutics with improved gastrointestinal stability

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

Humanized Mice for the Generation of HIV-1 Human Monoclonal Antibodies

Background: Despite the length of time HIV has been wreaking havoc on its victims, improvements in the prevention and treatment of HIV are needed. Anti-retroviral therapy can be effective but is expensive and not entirely accessible for people infected in third world countries. Several promising broadly neutralizing antibodies have been isolated from infected individuals; we propose that generating antigen specific human monoclonal antibodies using humanized mice further represents a promising approach to engineer prophylactic antibodies to reduce spread and infection of HIV. Methods: Immunodeficient mice were engrafted with fetal liver and thymus (BLT) prior to infection with different HIV isolates. HIV infection of the mice was monitored by viral load and antibody response followed by ELISA using gp120, gp41 or gp120/CD4 complex as antigens. Approximately 8-12 weeks post infection, spleens were harvested and splenocytes fused with human fusion partner HMMA 2.5 to isolate antibody-expressing hybridomas. Lead clones were scaled and purified for testing in functional assays such as TZM-bl neutralization assays as well as ADCVI to determine neutralizing and cytotoxic ability of the antibodies. Antibody sequences were also determined for analysis. Results: A robust, specific antibody response, of both IgG and IgA isotypes, was generated in response to HIV infection. Over 60 hybridomas were created that were not only immunoreactive with env antigens, but also had neutralization activity. Moreover, variable family usage was not limited and somatic mutation was clearly evident. Conclusions: These findings suggest that humanized BLT mice are a novel source for well-characterized, stable human monoclonal antibodies to HIV

Analytical characterization and formulation assessment of model secretory- immunoglobulin-A (sIgAs) for their potential use as low cost, orally delivered sIgAs

Enterotoxigenic Escherichia coli (ETEC) is a major cause of bacterial diarrheal disease in developing countries, especially among children and infants. ETEC is estimated to cause 280-400 million diarrheal episodes per year in children \u3c5 years of age, resulting in 300,000 to 500,000 deaths.1 Despite the need for a vaccine, there are currently no licensed vaccines against ETEC. Alternatively, passive immunization by oral delivery of pathogen-specific immunoglobulins is another promising approach to provide “instant” protection against ETEC. The potential advantages of oral delivery are reduced cost, simplicity of administration and localized treatment within the GI tract. Secretory IgA (sIgA) is of particular interest because it is naturally found in the mucosal surfaces within the GI tract, relatively more resistant to proteolysis by digestive enzymes (vs. IgGs), and can protect against enteric bacteria by directly neutralizing virulence factors.2 One major challenge of this approach is the instability of protein molecules during oral delivery (in the digestive tract) as well as during long-term storage (in various formulations). In this study, two proteins, sIgA1 and sIgA2 against heat labile toxin (LT, one of the major virulence factors of ETEC), were used as model sIgA molecules for developing analytical techniques and assessing stability (physicochemical as well as in vitro binding) under various conditions. A combination of biochemical and biophysical methods were employed to comprehensively characterize the sIgA1 and sIgA2 model proteins including primary structure, post translational modifications (i.e., N-linked glycans), size, apparent solubility, higher order structure and conformational stability as well as in vitro antigen binding. Using these characterization and stability indicating methods, we are monitoring the stability of these two model sIgAs both in an in vitro digestion model (to mimic in vivo degradation conditions), and during accelerated stability studies (to assess storage stability). Our goal is to use the information gained by these aforementioned methods and stability studies to design stable, low-cost liquid formulations for oral delivery of sIgAs in the developing world. Please click Additional Files below to see the full abstract

Preformulation Characterization and Stability Assessments of Secretory IgA Monoclonal Antibodies as Potential Candidates for Passive Immunization by Oral Administration

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease in children in developing countries, and there are no licensed vaccines to protect against ETEC. Passive immunization by oral delivery of ETEC-specific secretory IgAs (sIgAs) could potentially provide an alternative approach for protection in targeted populations. In this study, a series of physiochemical techniques and an in vitro gastric digestion model were used to characterize and compare key structural attributes and stability profiles of three anti-heat labile enterotoxin monoclonal antibodies (sIgA1, sIgA2 and IgG1 produced in CHO cells). The mAbs were evaluated in terms of primary structure, N-linked glycan profiles, size and aggregate content, relative apparent solubility, conformational stability, and in vitro antigen binding. Compared to IgG1 mAb, sIgA1 and sIgA2 mAbs showed increased sample heterogeneity, especially in terms of N-glycan composition and the presence of higher molecular weight species. The sIgA mAbs showed overall better physical stability and were more resistant to loss of antigen binding activity during incubation at low pH, 37 degrees C with pepsin. These results are discussed in terms of future challenges to design stable, low-cost formulations of sIgA mAbs as an oral supplement for passive immunization to protect against enteric diseases in the developing world

Discovery and Development of Human Monoclonal Antibodies to Block RhD Alloimmunization During Pregnancy

Exposure of an Rh negative mother to red blood cells (RBCs) of an Rh positive fetus results in alloimmunization and development of anti-RhD antibodies. The anti-RhD antibodies cause hemolytic disease of the new born babies during subsequent pregnancies. Current prophylactic treatment involves polyclonal anti-RhD IgG purified from plasma of humans and is administered in approximately 20% of pregnancies. While the current prophylaxis is effective, it involves the use of human plasma and non-RhD specific antibodies, thus posing a risk of transmitting infections and undesired antibody reactions. Moreover, there is a serious scarcity of plasma donors to meet the requirement of anti-RhD antibodies. In this study we propose to discover and develop anti-RhD monoclonal human antibodies to replace the current polyclonal prophylaxis. We are using humanized BLT mice (fetal CD34+ stem cells, liver and thymus) reconstituted with RhD negative donor material and were immunized by using adenovirus containing RhD transgene. Serum samples were collected after 4-6 weeks of immunization. Our results show that the RhD immunized mice had considerably higher titer of IgG and IgA antibodies in the serum compared to the control, suggesting an immune response developed upon immunization. Splenocytes from antibody producing mice will be fused with a human fusion partner for the isolation of hybridomas producing human monoclonal antibodies. The immunoreactivity and functional activity of these antibodies will be discussed

Immune features that afford protection from clinical disease versus sterilizing immunity to Bordetella pertussis infection in a nonhuman primate model of whooping cough

The respiratory bacterial infection caused by Bordetella pertussis (whooping cough) is the only vaccine-preventable disease whose incidence has been increasing over the last 3 decades. To better understand the resurgence of this infection, a baboon animal model of pertussis infection has been developed. Naïve baboons that recover from experimental pertussis infection are resistant both to clinical disease and to airway colonization when re-challenged. In contrast, animals vaccinated with acellular pertussis vaccine and experimentally challenged do not develop disease, but airways remain colonized for 4-6 weeks. We explored the possibility that the IgG antibody response to pertussis infection is qualitatively different from antibodies induced by acellular pertussis vaccination. IgG was purified from pertussis-convalescent baboons shown to be resistant to pertussis disease and airway colonization. Purified IgG contained high titers to pertussis toxin, pertactin, and filamentous hemagglutinin. This pertussis-immune IgG or control IgG was passively transferred to naïve, juvenile baboons before experimental airway pertussis inoculation. The control animal that received normal IgG developed a typical symptomatic infection including leukocytosis, cough and airway colonization for 4 weeks. In contrast, baboons that received convalescent IgG maintained normal WBC counts and were asymptomatic. However, despite remaining asymptomatic, their airways were colonized for 4-6 weeks with B. pertussis. All animals developed IgG and IgA anti-pertussis antibody responses. Interestingly, the clearance of B. pertussis from airways coincided with the emergence of a serum anti-pertussis IgA response. These studies demonstrate that passive administration of pertussis-specific IgG from previously infected animals can prevent clinical disease but does not affect prolonged airway colonization with B. pertussis. This outcome is similar to that observed following acellular pertussis vaccination. Understanding immune mechanisms—other than IgG—that are capable of preventing airway colonization with B. pertussis will be critical for developing more effective vaccines to prevent whooping cough