Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.     

Reformatting palivizumab and motavizumab from IgG to human IgA impairs their efficacy against RSV infection in vitro and in vivo

Respiratory syncytial virus (RSV) infection is a leading cause of hospitalization and mortality in young children. Protective therapy options are limited. Currently, palivizumab, a monoclonal IgG1 antibody, is the only licensed drug for RSV prophylaxis, although other IgG antibody candidates are being evaluated. However, at the respiratory mucosa, IgA antibodies are most abundant and act as the first line of defense against invading pathogens. Therefore, it would be logical to explore the potential of recombinant human IgA antibodies to protect against viral respiratory infection, but very little research on the topic has been published. Moreover, it is unknown whether human antibodies of the IgA isotype are better suited than those of the IgG isotype as antiviral drugs to combat respiratory infections. To address this, we generated various human IgA antibody formats of palivizumab and motavizumab, two well-characterized human IgG1 anti-RSV antibodies. We evaluated their efficacy to prevent RSV infection in vitro and in vivo and found similar, but somewhat decreased efficacy for different IgA subclasses and formats. Thus, reformatting palivizumab or motavizumab into IgA reduces the antiviral potency of either antibody. Moreover, our results indicate that the efficacy of intranasal IgA prophylaxis against RSV infection in human FcαRI transgenic mice is independent of Fc receptor expression.     

Epidemiological study of zoonoses derived from humans in captive chimpanzees

Emerging infectious diseases (EIDs) in wildlife are major threats both to human health and to biodiversity conservation. An estimated 71.8 % of zoonotic EID events are caused by pathogens in wildlife and the incidence of such diseases is increasing significantly in humans. In addition, human diseases are starting to infect wildlife, especially non-human primates. The chimpanzee is an endangered species that is threatened by human activity such as deforestation, poaching, and human disease transmission. Recently, several respiratory disease outbreaks that are suspected of having been transmitted by humans have been reported in wild chimpanzees. Therefore, we need to study zoonotic pathogens that can threaten captive chimpanzees in primate research institutes. Serological surveillance is one of several methods used to reveal infection history. We examined serum from 14 captive chimpanzees in Japanese primate research institutes for antibodies against 62 human pathogens and 1 chimpanzee-borne infectious disease. Antibodies tested positive against 29 pathogens at high or low prevalence in the chimpanzees. These results suggest that the proportions of human-borne infections may reflect the chimpanzee’s history, management system in the institute, or regional epidemics. Furthermore, captive chimpanzees are highly susceptible to human pathogens, and their induced antibodies reveal not only their history of infection, but also the possibility of protection against human pathogens.     

Overview of vaccines and vaccination

Of the 80-plus known infectious agents pathogenic for humans, there are now more than 30 vaccines against 26 mainly viral and bacterial infections and these greatly minimize subsequent disease and prevent death after exposure to those agents. This article describes the nature of the vaccines, from live attenuated agents to subunits, their efficacy and safety, and the kind of the immune responses generated by those vaccines, which are so effective. To date, all licensed vaccines generate especially specific antibodies, which attach to the infectious agent and therefore can very largely prevent infection. These vaccines have been so effective in developed countries in preventing mortality after a subsequent infection that attempts are being made to develop vaccines against many of the remaining infectious agents. Many of the latter are difficult to manipulate; they can cause persisting infections or show great antigenic variation. A range of new approaches to improve selected immune responses, such as immunization with DNA or chimeric live vectors, viral or bacterial, are under intense scrutiny, as well as genomic analysis of the agent.     

Antigenic analysis of equine infectious anemia virus (EIAV) variants by using monoclonal antibodies: epitopes of glycoprotein gp90 of EIAV stimulate neutralizing antibodies.

Monoclonal antibodies produced against the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV), a lentivirus, were studied for reactivity with the homologous prototype and 16 heterologous isolates. Eighteen hybridomas producing monoclonal antibodies (MAbs) were isolated. Western blot (immunoblot) analyses indicated that 10 were specific for the major envelope glycoprotein (gp90) and 8 for the transmembrane glycoprotein (gp45). Four MAbs specific to epitopes of gp90 neutralized prototype EIAV infectivity. These neutralizing MAbs apparently reacted with variable regions of the envelope gp90, as evidenced by their unique reactivity with the panel of isolates, suggesting recognition of at least three different neutralization epitopes. The conformation of these epitopes appears to be continuous, as they resisted treatment with sodium dodecyl sulfate and reducing reagents. Monoclonal antibodies that reacted with conserved epitopes on gp90 or gp45 failed to neutralize EIAV. Our data also demonstrated that there was a large spectrum of possible EIAV serotypes and confirmed that antigenic variation occurs with high frequency in EIAV. Moreover, the data showed that variation is a rapid and random process, as no pattern of variant evolution was evident by comparison of 13 isolates from parallel infections. These results represent the first production of neutralizing MAbs specific for a lentivirus glycoprotein and document alterations in one or more neutralization epitopes of the major surface glycoprotein among sequential isolates of EIAV recovered during persistent infection.     

New immunotherapeutic strategies to control vaginal candidiasis

The widespread occurrence of mucosal infections caused by Candida, in particular recurrent vulvovaginal candidiasis among fertile-age women, together with the paucity of safe candidacidal antimycotics, have prompted a great number of investigations into the immunotherapy of candidal vaginitis. This article will discuss three different experimental approaches demonstrated to be potentially transferable to human disease: (1) the use of antibodies against well-defined cell-surface adhesins or enzymes; (2) the generation of yeast killer-toxin-like candidacidal anti-idiotypic antibodies and their engineered molecular derivatives (e.g. single chains, peptides); and (3) the generation of therapeutic vaccines and immunomodulators.     

Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.     

Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.     

Live bivalent vaccine for parainfluenza and influenza virus infections

Influenza and human parainfluenza virus infections are of both medical and economical importance. Currently, inactivated vaccines provide suboptimal protection against influenza, and vaccines for human parainfluenza virus infection are not available, underscoring the need for new vaccines against these respiratory diseases. Furthermore, to reduce the burden of vaccination, the development of multivalent vaccines is highly desirable. Thus, to devise a single vaccine that would elicit immune responses against both influenza and parainfluenza viruses, we used reverse genetics to generate an influenza A virus that possesses the coding region for the hemagglutinin/neuraminidase ectodomain of parainfluenza virus instead of the influenza virus neuraminidase. The recombinant virus grew efficiently in eggs but was attenuated in mice. When intranasally immunized with the recombinant vaccine, all mice developed antibodies against both influenza and parainfluenza viruses and survived an otherwise lethal challenge with either of these viruses. This live bivalent vaccine has obvious advantages over combination vaccines, and its method of generation could, in principle, be applied in the development of a "cocktail" vaccine with efficacy against several different infectious diseases.     

In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein.

Six different anti-HIV envelope antibodies and one irrelevant control antibody were coupled to ricin A chain and tested for their efficacy in inhibiting HIV tissue culture infections. The anti-HIV antibodies consisted of five monoclonals, three of murine and two of human origin, and one polyclonal preparation prepared by affinity purifying pooled serum antibodies from HIV-infected humans on rgp160. The binding specificity of the antibodies was defined by ELISA by using recombinant envelope proteins and synthetic peptides, and by flow cytometry on HIV-infected cells. The in vitro efficacy of the antibodies was tested by the abilities of the immunotoxins to inhibit protein synthesis in persistently infected cell lines and by their abilities to inhibit HIV production during both acute and persistent infection as measured with an HIV-specific focal immunoassay. The immunotoxins were tested against a panel of distinctly different HIV isolates. The results indicate the following: 1) A mAb to the immunodominant neutralizing loop was highly effective against homologous strains of HIV, but had no activity against heterologous HIV. 2) The efficacy of anti-gp41 mAb varied depending upon the epitope recognized and possibly the affinity of binding to gp41. 3) The polyclonal human anti-gp160 antibodies produced the immunotoxin with the broadest specificity for different HIV strains and the greatest specific activity. This is related to the polyclonal nature of the preparation rather than an increase in relative avidity of the antibody. 4) Activity of an immunotoxin is not a direct function of the binding of the antibody to the surface of infected cells. 5) The ability of an immunotoxin to halt the spread of infection through a tissue culture cell population is dependent upon the ability of the antibody to neutralize the virus as well as the activity of the toxin. Our data suggest that efficacious immunotoxins for the treatment of AIDS may be made with polyclonal anti-envelope antibodies derived from the serum of patients who have been infected with HIV or with appropriately chosen anti-gp41 antibodies.     

Determinants of the efficacy of viro-immunotherapy: A review

Oncolytic virus immunotherapy is rapidly gaining interest in the field of immunotherapy against cancer. The minimal toxicity upon treatment and the dual activity of direct oncolysis and immune activation make therapy with oncolytic viruses (OVs) an interesting treatment modality. The safety and efficacy of several OVs have been assessed in clinical trials and, so far, the Food and Drug Administration (FDA) has approved one OV. Unfortunately, most treatments with OVs have shown suboptimal responses in clinical trials, while they appeared more promising in preclinical studies, with tumours reducing after immune cell influx. In several clinical trials with OVs, parameters such as virus replication, virus-specific antibodies, systemic immune responses, immune cell influx into tumours and tumour-specific antibodies have been studied as predictors or correlates of therapy efficacy. In this review, these studies are summarized to improve our understanding of the determinants of the efficacy of OV therapies in humans and to provide insights for future developments in the viro-immunotherapy treatment field.     

Active and passive immunization with rHyr1p-N protects mice against hematogenously disseminated candidiasis

We previously reported that Candida albicans cell surface protein Hyr1 encodes a phagocyte killing resistance factor and active vaccination with a recombinant N-terminus of Hyr1 protein (rHyr1p-N), significantly protects immunocompetent mice from disseminated candidiasis. Here we report the marked efficacy of rHyr1p-N vaccine on improving the survival and reducing the fungal burden of disseminated candidiasis in both immunocompetent and immunocompromised mice using the FDA-approved adjuvant, alum. Importantly, we also show that pooled rabbit anti-Hyr1p polyclonal antibodies raised against 8 different peptide regions of rHyr1p-N protected mice in a hematogenously disseminated candidiasis model, raising the possibility of developing a successful passive immunotherapy strategy to treat this disease. Our data suggest that the rabbit anti-Hyr1p antibodies directly neutralized the Hyr1p virulence function, rather than enhanced opsonophagocytosis for subsequent killing by neutrophil in vitro. Finally, the rHyr1p-N vaccine was protective against non-albicans Candida spp. These preclinical data demonstrate that rHyr1p-N is likely to be a novel target for developing both active and passive immunization strategies against Candida infections.     

噬菌体特异性抗体对噬菌体治疗的影响

噬菌体治疗已成为当下防控泛耐药细菌感染的重要选择。噬菌体作为含有蛋白和核酸组分的病毒颗粒,经不同途径进入机体后,均能诱导机体产生特异性中和抗体。本文就噬菌体治疗过程中诱导机体产生的特异性中和抗体、抗体的产生规律、抗体是否影响噬菌体治疗疗效,以及可能克服抗体影响噬菌体治疗的方法等进行论述。噬菌体颗粒诱导特异性中和抗体的产生及血清抗噬菌体活性的水平与噬菌体的给予途径、类型和剂量、结构蛋白以及宿主的免疫状态、感染部位、治疗持续时间等均有关,且不同类型抗体产生时间和强度不同,均能中和噬菌体从而降低其杀菌效果。这提示在使用噬菌体治疗耐药细菌感染时,需要探索克服噬菌体中和抗体干扰的方法,或针对机体不同状态及感染类别制定相应的治疗策略,降低诱导机体产生噬菌体特异性中和抗体的风险,以获得最佳治疗效果。     

Characterization of neutralizing mouse‐human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse‐human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V_H) and light chains (V_L) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO‐DG44 cells were transfected with these plasmids and mouse‐human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V_H or V_L domains were constructed. A shuffling antibody (AC2494) that derived its V_H and V_L domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high‐potency neutralizing chimeric antibodies by selecting and shuffling V_H and V_L domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.     

Recent developments in new and old viral infections

Until recently, concerns regarding viral infections and hematologic malignancies were primarily focused on the transplantation of an allogeneic graft. In the last years, the use of immunomodulatory agents such as monoclonal antibodies (e.g. anti CD20, anti CD52) directed against lymphocyte antigens in the treatment of hematopoietic malignancies (e.g. lymphoma, chronic lymphocytic leukemia) has added a great potential to impact on the incidence, severity and timing of viral infections. Patients may acquire viral infections through several mechanisms including transfusion, community exposure or via the donor origin in the case of stem cell transplant. Endogenous reactivation of latent viruses is also commonly observed. Viral replication may lead directly to viral diseases or induce indirect effects such as increased incidence of opportunistic infections and decreased patient survival. Traditionally, herpesviruses have been and are still today the most important viruses in patients with hematologic malignancies. Nowadays, several emerging viral infections have been highlighted as being of concern in this patients' population.     

Protective efficacy of IgA monoclonal antibodies to O and H antigens in a mouse model of intranasal challenge with Salmonella enterica serotype Enteritidis

Protective properties of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens of Salmonella enterica serotype Enteritidis (S. enteritidis) were evaluated in a model of generalized infection after intranasal (i.n.) inoculation of BALB/c mice. Passive i.n. instillation of antibodies 1 h before i.n. challenge did not prevent infection, and mice developed rapid inflammatory response in the lower respiratory tract. The passive systemic immunization was partially protective and a single intravenous (i.v.) injection of both O and H antigen specific IgA antibodies prolonged survival period of the infected animals. Permanent secretion of O:9 specific IgA MAb 177E6 into the respiratory tract in a "backpack" tumor model protected 50% of animals infected i.n. with a high dose of virulent S. enteritidis strain. Thus, secretory IgA (S-IgA) directed against O:9 antigen alone can prevent bacterial invasion in the respiratory epithelium.     

Human cytomegalovirus infection elicits a glycoprotein M (gM)/gN-specific virus-neutralizing antibody response

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen that infects 40 to 90% of adult human populations. HCMV infections are often asymptomatic in healthy individuals but can cause severe organ and life-threatening disease in immunocompromised patients. The antiviral antibody response to HCMV infection is complex and is known to include virus-neutralizing antibody production against surface glycoproteins encoded by HCMV. We have investigated the human antibody response to a complex of HCMV surface glycoproteins composed of glycoprotein M (gM)/gN, the gene products of the UL100 and UL73 open reading frames. Mouse monoclonal antibodies generated against gM/gN have previously been shown to neutralize HCMV infection of human fibroblasts in vitro. To determine whether human antibodies reactive with the gM/gN complex possess virus-neutralizing properties, we isolated human antibodies reactive with gM/gN from pooled human HCMV hyperimmune globulin by affinity purification using recombinant gM/gN. The affinity-purified human anti-gM/gN antibodies reacted specifically by immunofluorescence with HCMV-infected human fibroblasts and with cells transiently expressing gM/gN, but not with cells transfected with plasmids encoding other immunogenic HCMV proteins. The anti-gM/gN antibodies also reacted specifically only with gM/gN in immunoblot assays using lysates of transfected cells expressing specific HCMV proteins. Last, human anti-gM/gN antibodies efficiently neutralized infectious HCMV in vitro with a capacity comparable to that of human anti-gB antibodies. These data indicated that gM/gN can elicit a virus-neutralizing antibody response in humans infected with HCMV and therefore should be considered a potential candidate for inclusion in prophylactic CMV vaccines.     

Potential therapeutic use of antibodies directed towards HuIFN-γ

IFN-γ is an important regulator of immune responses and inflammation. Studies in animal models of inflammation, autoimmunity, cancer, transplant rejection and delayed-type hypersensitivity have indicated that administration of antibodies against IFN-γ can prevent the occurrence of diseases or alleviate disease manifestations. Therefore, it is speculated that such antibodies may have therapeutical efficacy in human diseases. Since animal-derived antibodies are immunogenic in patients several strategies are being developed in order to reduce or abolish this human anti-mouse antibody (HAMA) response. In our laboratory, we have constructed a single-chain variable fragment (scFv) derived from a mouse antibody with neutralizing potential for human IFN-γ. A scFv consists of only variable domains tethered together by a flexible linker. The scFv was demonstrated to neutralize the antiviral activity of HuIFN-γin vitro and therefore might be considered as a candidate for human therapy.