A recombinant metalloprotease antigen of Vibrio vulnificus elicits protective antibodies in a murine model

Aims: To investigate whether Vibrio vulnificus metalloprotease (VvpE) can induce the production of specific anti‐VvpE antibody to confer effective protection against Vibrio vulnificus infection and to evaluate the possibility of VvpE as a potential vaccine candidate against disease caused by V. vulnificus. Methods and Results: The gene encoding the 65‐kDa VvpE of V. vulnificus was amplified by PCR and cloned into the expression vector pET21(b). The recombinant VvpE of V. vulnificus was expressed in Escherichia coli BL21(DE3). This His₆‐tagged VvpE was purified and injected intramuscularly into mice to evaluate its ability to stimulate immune response. Specific antibody levels were measured by ELISA. The 75% protective efficacy of recombinant VvpE was evaluated by active immunization and intraperitoneal challenge with V. vulnificus in mice. Conclusions: The recombinant His₆‐tagged VvpE of V. vulnificus is capable of inducing high antibody response in mice to confer effective protection against lethal challenge with V. vulnificus. VvpE might be a potential vaccine candidate to against V. vulnificus infection. Significance and Impact of the Study: This study uses His₆‐tagged VvpE to act as vaccine that successfully induces effective and specific anti‐VvpE antibody and offers an option for the potential vaccine candidate against V. vulnificus infection.     

Strategies against human papillomavirus infection and cervical cancer

Papillomaviruses infect a wide variety of animals, including humans. The human papillomavirus (HPV), in particular, is one of the most common causes of sexually transmitted disease. More than 200 types of HPV have been identified by DNA sequence data, and 85 HPV genotypes have been well characterized to date. HPV can infect the basal epithelial cells of the skin or inner tissue linings, and are, accordingly, categorized as either cutaneous or mucosal type. HPV is associated with a panoply of clinical conditions, ranging from innocuous lesions to cervical cancer. In the early 1980s, studies first reported a link between cervical cancer and genital HPV infection. Genital HPV infections are now recognized to be a major risk factor in at least 95% of cervical cancers. 30 different HPV genotypes have been identified as causative of sexually transmitted diseases, most of which induce lesions in the cervix, vagina, vulva, penis, and anus, as the result of sexual contact. There is also direct evidence demonstrating that at least four of these genotypes are prerequisite factors in cervical cancer. The main aim of this review was to evaluate the current literature regarding the pathovirology, diagnostics, vaccines, therapy, risk groups, and further therapeutic directions for HPV infections. In addition, we reviewed the current status of HPV infections in South Korean women, as evidenced by our data.     

Further studies on an intermediate host murine model showing that a primary Echinococcus granulosus infection is protective against subsequent oncospheral challenge

We describe the use of a murine model to evaluate resistance against subsequent challenge following a primary infection with oncospheres of Echinococcus granulosus. Mice (Kunming strain) were infected with hatched oncospheres of Echinococcus granulosus; 21 days later a second challenge was given by a different route of infection. A primary infection by intraperitoneal (i.p.) injection stimulated 100 and 90.5% protection in terms of reduced cyst numbers against a secondary infection given subcutaneously (s.c.) or intravenously (i.v.), respectively. A primary infection given s.c. followed by i.p. or i.v. challenge resulted in 84.0 and 100% protection, respectively. Intravenous infection followed by i.p. or s.c. challenge resulted in 98.5 and 69.4% protection, respectively. With the i.v. route of infection, almost all resultant cysts were present in the lungs. The data show that a primary infection with oncospheres can induce total or a high degree of protection against a subsequent challenge and confirms that natural (concomitant) immunity can be stimulated in the intermediate host as the result of a primary infection. This may explain the decline in hydatid infection in sheep older than 2 years in hyper-endemic areas such as those found in Xingjiang, China. These older sheep may have been earlier infected and have subsequently self-cured, with the primary infection stimulating an immune response that protects the intermediate host animals from further infection.     

Characterization of murine polyclonal antisera and monoclonal antibodies generated against intact and denatured human papillomavirus type 1 virions.

Human papillomavirus type 1 (HPV1) virions, both as intact virion particles (IVP) and as detergent-denatured virions (DDV), were used to prepare polyclonal antisera and monoclonal antibodies (MAbs) in BALB/c mice. Anti-IVP antiserum contained type-specific HPV1 L2-reactive antibodies and no detectable HPV1 L1-reactive antibodies. Anti-IVP MAbs recognized a linear epitope between L2 amino acids 102 and 108 (PIDVVDP). Anti-DDV antiserum contained type-specific HPV1 L1-reactive and HPV1 L2-reactive antibodies. An anti-DDV MAb recognized a linear epitope between L1 amino acids 127 and 133 (AENPTNY). HPV1a L1- and L2-encoded polypeptides expressed in Saccharomyces cerevisiae and by in vitro translation were equivalent in size to the major and minor virion capsid proteins, respectively.     

Polyclonal and specific antibodies mediate protective immunity against enteric helminth infection

Anti-helminth immunity involves CD4+ T cells, yet the precise effector mechanisms responsible for parasite killing or expulsion remain elusive. We now report an essential role for antibodies in mediating immunity against the enteric helminth Heligmosomoides polygyrus (Hp), a natural murine parasite that establishes chronic infection. Polyclonal IgG antibodies, present in naive mice and produced following Hp infection, functioned to limit egg production by adult parasites. Comparatively, affinity-matured parasite-specific IgG and IgA antibodies that developed only after multiple infections were required to prevent adult worm development. These data reveal complementary roles for polyclonal and affinity-matured parasite-specific antibodies in preventing enteric helminth infection by limiting parasite fecundity and providing immune protection against reinfection, respectively. We propose that parasite-induced polyclonal antibodies play a dual role, whereby the parasite is allowed to establish chronicity, while parasite load and spread are limited, likely reflecting the long coevolution of helminth parasites with their hosts.     

Immunization with major outer membrane protein of Vibrio vulnificus elicits protective antibodies in a murine model

Sera from rabbits were infected with Vibrio vulnificus containing an antibody against major outer membrane protein (MOMP). MOMP of V. vulnificus ATCC 27562 were isolated and purified by Sarkosyl and TritonX-100 dual treatment. Molecular size of MOMP was identified as 36-kDa on 13% SDS-PAGE. The sequence of the first 26 amino acid residues from the N-terminal end of the protein is AELYNQDGTSLDMGGRAEARLSMKDG , which is a perfect match with OmpU of V. vulnificus CMCP6 and YJ016. MOMP specific IgM and IgG were investigated in groups of mice. The group of mice immunized with MOMP and Alum showed higher levels of IgG2b than the group immunized with only MOMP. Vaccination with MOMP resulted in protective antibodies in the mouse infection experiment.     

Carrageenan is a potent inhibitor of papillomavirus infection

Certain sexually transmitted human papillomavirus (HPV) types are causally associated with the development of cervical cancer. Our recent development of high-titer HPV pseudoviruses has made it possible to perform high-throughput in vitro screens to identify HPV infection inhibitors. Comparison of a variety of compounds revealed that carrageenan, a type of sulfated polysaccharide extracted from red algae, is an extremely potent infection inhibitor for a broad range of sexually transmitted HPVs. Although carrageenan can inhibit herpes simplex viruses and some strains of HIV in vitro, genital HPVs are about a thousand-fold more susceptible, with 50% inhibitory doses in the low ng/ml range. Carrageenan acts primarily by preventing the binding of HPV virions to cells. This finding is consistent with the fact that carrageenan resembles heparan sulfate, an HPV cell-attachment factor. However, carrageenan is three orders of magnitude more potent than heparin, a form of cell-free heparan sulfate that has been regarded as a highly effective model HPV inhibitor. Carrageenan can also block HPV infection through a second, postattachment heparan sulfate-independent effect. Carrageenan is in widespread commercial use as a thickener in a variety of cosmetic and food products, ranging from sexual lubricants to infant feeding formulas. Some of these products block HPV infectivity in vitro, even when diluted a million-fold. Clinical trials are needed to determine whether carrageenan-based products are effective as topical microbicides against genital HPVs.     

An antigen chimera of poliovirus induces antibodies against human papillomavirus type 16.

It has been established that the surface of poliovirus type 1 can be extensively modified to incorporate antigenic domains from other poliovirus serotypes and from unrelated viruses. The fact that the modified (chimeric) viruses exhibit dual antigenicity and immunogenicity led us to explore the possibility of using the Sabin vaccine strain of poliovirus type 1 as a vector for the presentation of antigenic domains from human papillomavirus type 16 (HPV-16), a virus associated with the development of cervical carcinoma. We report here the construction and characterization of a chimeric poliovirus containing a 16-residue sequence derived from the major capsid protein (L1) of HPV-16. This virus chimera stimulated the production in rabbits of antibodies which recognized the HPV-16-derived peptide and an L1 fusion protein synthesized in Escherichia coli and detected HPV-16 in human biopsy material by immunoperoxidase staining. The possibility that poliovirus-HPV chimeras could be used as vaccines against HPV-16 is discussed.     

A protective and broadly cross-neutralizing epitope of human papillomavirus L2

We generated a monoclonal antibody, RG-1, that binds to highly conserved L2 residues 17 to 36 and neutralizes human papillomavirus 16 (HPV16) and HPV18. Passive immunotherapy with RG-1 was protective in mice. Antiserum to the HPV16 L2 peptide comprising residues 17 to 36 (peptide 17-36) neutralized pseudoviruses HPV5, HPV6, HPV16, HPV 18, HPV31, HPV 45, HPV 52, HPV 58, bovine papillomavirus 1, and HPV11 native virions. Depletion of HPV16 L2 peptide 17-36-reactive antibodies from cross-neutralizing rabbit and human L2-specific sera abolished cross-neutralization and drastically reduced neutralization of the cognate type. This cross-neutralization of diverse HPVs associated with cervical cancer, genital warts, and epidermodysplasia verruciformis suggests the possibility of a broadly protective, peptide-based vaccine.     

Chimeric papillomavirus virus-like particles induce a murine self-antigen-specific protective and therapeutic antitumor immune response

The use of chimeric virus-like particles represents a new strategy for delivering tumor antigens to the immune system for the initiation of antitumor immune responses. Immunization of DBA/2 mice with the P1A peptide derived from the P815 tumor-associated antigen P1A induced specific T-cell tolerance, resulting in progression of a regressor P815 cell line in all animals. However, immunization with a human papillomavirus type 16 L1 virus-like particle containing the P1A peptide in the absence of adjuvant induced a protective immune response in mice against a lethal tumor challenge with a progressor P815 tumor cell line. Additionally, we demonstrated that these chimeric virus-like particles could be used therapeutically to suppress the growth of established tumors, resulting in a significant survival advantage for chimeric virus-like particle-treated mice compared with untreated control mice. Chimeric virus-like particles can thus be used as a universal delivery vehicle for both tolerizing and antigenic peptides to induce a strong protective and therapeutic antigen-specific antitumor immune response.     

A model of enhancement and inhibition of HIV infection of monocytes by antibodies against HIV

Antibodies (Ab) specific for epitopes on HIV glycoprotein gp120 or gp41 can inhibit or enhance HIV infection of human cellsin vitro. These effects may have significant implications both for the pathogenesis of chronic HIV infection and for vaccine development. A particularly puzzling findingin vitro is antibody dependent enhancement (ADE) at low concentrations of Ab while high concentrations of the same antibody inhibit infection. Similar phenomena have been observed for other enveloped viruses. Antibodies can inhibit infection by several mechanisms. However, by binding to receptors on target cells, virus bound antibodies can also enhance adhesion to these cells and thereby facilitate infection. We propose a mathematical model that describes how these two processes interact and hereby provide an explanation for the observed enhancement/neutralization phenomena. Simulation results were validated with good agreement against empirical data from antibody dependent enhancement of HIV infection of monocytoid (U937) cellsin vitro, and the model should be applicable to otherin vitro systems involving different cells and viruses.The model indicates that for the common type of HIV neutralizing antibody, acting at a post-CD4-binding step, there is a neutralizing window defined by the affinity and concentration of antibody.     

Neutralizing antibodies against oncogenic human papillomavirus as a possible determinant of the fate of low-grade cervical intraepithelial neoplasia

To determine whether neutralizing antibodies (NAs) against HPV16 is responsible for a higher regression rate of low-grade cervical intraepithelial neoplasia (CIN1), we investigated an association between the presence of the NAs and the fate of the HPV16-related CIN1. All the women examined in this study had HPV16 positive cervix. The women were allocated into four groups by their cervical pathology, i.e., non-pathological (n:7), CIN1 (n:37), CIN2/3 (n:19), and cervical cancer (n:13). Their sera were tested for the presence of NAs against HPV16 by an in vitro assay using HPV16-pseudovirions. As for the CIN1 cases, clinical regression of the lesions were compared between NA-positive and NA-negative groups. Copy number of HPV16-DNA in smear samples was measured by quantitative PCR. The incidence of the presence of the NAs in the women with a non-pathological cervix (85.7%) was significantly higher than in the CIN1 cases (21.5%), the CIN2/3 cases (15.7%), and the cervical cancer cases (0%) (p<0.0001). The regression of the CIN1 lesion was closely associated with the presence of the N As (p=0.0002). The presence of the NAs was associated with low-level copy number of the viral DNA relative to the NA-negative group (p=0.05). The presence of the NAs against HPV16 was associated with a higher regression rate of HPV-related CIN1 lesions. The NAs seem to have a role in deterring HPV-related cervical lesions from progressing to CIN2/3 by inhibiting the infection with de novo replicated HPV. This study further suggests that HPV vaccine to induce the NAs may be effective in eliminating CIN lesions, especially in the NA-negative cases.     

IL-6 is required for protective immune responses against early filarial infection

IL-6 has a wide range of biological activities that includes anti- and pro-inflammatory aspects. In this study, we investigated the role of IL-6 in immune responses to the rodent filarial nematode Litomosoides sigmodontis, a model for human filarial infections. IL-6^?/? mice had a significantly increased worm burden after natural infection compared with wild type controls at early time points p.i. Given that the worm burden in IL-6^?/? mice was already increased at the time point the infective larvae reached the pleural cavity, immune responses that may facilitate the migration from the site of infection (skin) via the lymphatics to the pleural cavity were analysed. Increased vascular permeability may facilitate larval migration, but blocking of histamine receptors had no effect on worm burden and vascular permeability was similar between IL-6^?/? mice and wild type controls. In contrast, blocking mast cell degranulation reduced the worm burden in IL-6^?/? mice partially, suggesting that release of mast cell-derived mediators improves larval migration to some degree. Protective immune responses within the skin were involved, as bypassing the skin barrier by inoculating infective L3s subcutaneously resulted in a comparable worm recovery in both mouse strains. Analysis of the cellular composition by flow cytometry and PCR array in the skin after exposure to filarial extract or L3s, respectively, indicate that the absence of IL-6 results in a delayed recruitment of neutrophils and macrophages to the site of initial infection. These results demonstrate that IL-6 is essentially involved in protective immune responses within the skin that impair migration of infective L3s.     

Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins

The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis.     

Complement activation is required for induction of a protective antibody response against West Nile virus infection

Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.     

A prospective study of human papillomavirus infection of the cervix

In a prospective study 42 women, diagnosed as having low grade cervical intraepithelial neoplasia (CIN), made a total of 281 clinic visits over a 45 month period. At each visit, they were subjected to cytological and colposcopical examination and samples were taken for human papillomavirus (HPV) DNA hybridization studies and for the detection of non-HPV infections. HPV types 16 and/or 18 were found in 25% of all the samples tested and these virus types were detected in five of six (83%) women whose lesions progressed compared to seven of 14 (50%) of those whose lesions regressed. The presence of HPV DNA was not a good prognostic indicator of progression since half of those whose disease regressed also harboured these viruses at some time. The recording of non-HPV infections almost 10 times more often in the women whose disease regressed than in those whose disease progressed could probably be accounted for by the former having a larger number of follow-up visits. Nevertheless, the significance of non-HPV infections also remains unclear.     

Metallothionein is a crucial protective factor against Helicobacter pylori-induced gastric erosive lesions in a mouse model

Infection with the gastric pathogen Helicobacter pylori (H. pylori) causes chronic gastritis, peptic ulcer, and gastric adenocarcinoma. These diseases are associated with production of reactive oxygen species (ROS) from infiltrated macrophages and neutrophiles in inflammatory sites. Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that can act not only as a metal-binding protein, but also as a ROS scavenger. In the present study, we examined the role of MT in the protection against H. pylori-induced gastric injury using MT-null mice. Female MT-null and wild-type mice were challenged with H. pylori SS1 strain, and then histological changes were evaluated with the updated Sydney grading system at 17 and 21 wk after challenge. Although the colonization efficiency of H. pylori was essentially the same for MT-null and wild-type mice, the scores of activity of inflammatory cells were significantly higher in MT-null mice than in wild-type mice at 17 wk after challenge. Histopathological examination revealed erosive lesions accompanied by infiltration of inflammatory cells in the infected MT-null mice but not in wild-type mice. Furthermore, activation of NF-kappaB and expression of NF-kappaB-mediated chemokines such as macrophage inflammatory protein-1alpha and monocytes chemoattractant protein-1 in gastric cells were markedly higher in MT-null mice than in wild-type mice. These results suggest that MT in the gastric mucosa might play an important role in the protection against H. pylori-induced gastric ulceration.