The Leeuwenhoek lecture, 1985. A molecular biologist's view of viral hepatitis

Three forms of viral hepatitis can be distinguished serologically. Hepatitis A virus is a picornavirus, which is being studied increasingly after its propagation in cell cultures. The B virus (HBV) is the prototype of a family now termed hepadna viruses and is by far the best understood. The third, by exclusion, is non-A non-B, about which little else is known. Molecular cloning methods enable copies of viral genes to be propagated and analysed quite readily and provide the means for isolation and expression of individual genes in microbial and animal cells. Determination of the nucleotide sequences of HBV DNA revealed its genetic organization and so guided studies of the mechanism of gene expression both in infected animals and cultures of transformed cells. Replication of the viral genome has also been studied in natural infections, particularly with duck HBV, but also with the human virus. Expression of HBV genes in microbial cells is valuable as a source of antigens for diagnostic reagents and vaccine preparations, but has also been of consequence for the identification of viral gene products not previously recognized and which are of considerable current interest. The methods and materials now available provide additional opportunities for inquiring into the course of viral infection, replication of the virus and, for HBV, the possible role in the development of hepatomas of integration of the viral genome into the host chromosome.     

TLR3 plays significant roles against hepatitis B virus

Hepatitis B virus (HBV) as the main prevalent infectious agent, play important roles in inducing severe liver diseases. Previous studies demonstrated that during prolonged forms of hepatitis B infection including chronic, asymptomatic and occult forms, patients are unable to eradicate HBV from hepatocytes completely. The main mechanisms responsible for development of the forms of hepatitis B are yet to be identified. Investigators suggested that the various genetic and immunological parameters of the patients may are responsible for resulting in the prolonged infection forms. It has been evidenced that TLRs play key roles in inducing appropriate immune responses, against viral infections. Therefore, these molecules can be considered as crucial sensors for HBV detection to induce immune responses against this virus. It has also been documented that the TLR3 detects intracellular viral dsRNA and subsequently activates NF-κB via the TRIF pathway. Therefore, impaired TLR3 expression may result in inappropriate immune responses against HBV which is reported in prolonged forms of hepatitis B. This review collected the recent information regarding the important roles of TLR3 in immune responses against HBV and also the status of TLR3 expression and its genetic variations in prolonged forms of HBV infections.     

Cellular and molecular mechanisms of vaccine-induced protection against retroviral infections

More than 15 years after the discovery of human immunodeficiency virus (HIV), researchers are still struggling to design a protective AIDS vaccine. A remaining problem is a lack of basic knowledge about the immunological requirements for protection against retroviruses. Infection of macaque monkeys with simian immunodeficiency virus is still the best model for HIV vaccine research. However, in this model it remains difficult to determine protective immunological mechanisms because of limited numbers of experimental animals and their genetic heterogeneity. Thus, fundamental concepts in retroviral immunology have to be defined in other ways such as mouse models. This minireview summarizes new findings on cellular and molecular mechanisms in protection of mice against Friend murine retrovirus infection. It has been shown that complex immune responses, including B and T cell responses, are required for efficient protection in this model. Multiple viral antigens are necessary to elicit such broad immune reactivity. Efficacious vaccines must protect not only against acute disease, but also against the establishment of persistent infections or the host is at serious risk of virus reactivation. The minireview closes with a discussion on the relevance of findings from the mouse model on the design of a protective vaccine against HIV.     

Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen

There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.     

Chemoimmunotherapy of chronic hepatitis B virus infection in the woodchuck model overcomes immunologic tolerance and restores T-cell responses to pre-S and S regions of the viral envelope protein

Treatment of chronic hepatitis B virus (HBV) infection could combine potent antiviral drugs and therapeutic vaccines to overcome immunological tolerance and induce the recovery phenotype to protect against disease progression. Conventional vaccination of woodchucks chronically infected with the woodchuck hepatitis virus (WHV) elicited differential T-cell response profiles depending on whether or not carriers were treated with the potent antiviral drug clevudine (CLV), which significantly reduces viral and antigen loads. The differential T-cell responses defined both CLV-dependent and CLV-independent epitopes of the pre-S and S regions of the WHV envelope protein. Only combined treatment involving CLV and conventional vaccine therapeutically restored the T-cell response profile of chronic WHV carrier woodchucks to that seen in prophylactic vaccination and in recovery from acute WHV infection. The results have implications for mechanisms of immunological tolerance operating in chronic HBV infection and suggest that such combined chemoimmunotherapy may be useful for treatment of humans with chronic HBV infection.     

Primary prevention of hepatocellular carcinoma in developing countries

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world with 80% of cases occurring in developing countries. The cancer is rapidly fatal in almost all cases with survival generally less than 1 year from diagnosis. The major risk factors for this cancer have been identified as chronic infection with hepatitis B (HBV) and hepatitis C (HCV) viruses and dietary exposure to aflatoxins. There is a safe and effective vaccine to prevent chronic HBV infection. Given estimates that approximately 70% of HCC in developing countries is attributable to HBV then vaccination could prevent more than 250,000 cases per year in these areas of the world. A major challenge now is to ensure the availability of vaccine in countries with endemic infection. Development of a vaccine against HCV is more problematic due to the genetic heterogeniety of the virus. However, with 24% of HCC in developing countries attributable to HCV (approximately 93,000 cases per year) a vaccine would make a major contribution to cancer prevention. Aflatoxins contaminate dietary staple foods (groundnuts, maize), are potent animal hepatocarcinogens and are carcinogenic in humans with particularly high risks in individuals with a concomitant infection with HBV. Reduction of exposure can be addressed at the community level either pre- or post-harvest by limiting fungal contamination of crops; approaches may involve low technology post-harvest measures to limit fungal growth or genetic engineering of crops to be resistant to fungal infection or toxin biosynthesis. An alternative measure is to modulate the metabolism of aflatoxins once ingested using chemopreventive agents e.g., oltipraz. The resources available in countries with endemic hepatitis infection and fungal contamination of foods are often severely limited. Clearly HBV vaccination has to be the priority in the reducing the incidence of HCC. However, there are currently 360 million chronic HBV carriers worldwide and HBV vaccine is still not incorporated into many national immunisation programs. Thus measures to reduce food spoilage by fungi and the associated dietary exposure to aflatoxins is also a desirable public health goal.     

Bicistronic DNA Vaccines Simultaneously Encoding HIV,HSV and HPV Antigens Promote CD8+ T Cell Responses and Protective Immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.     

Expression and Characterization of Recombinant,Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments.The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).     

The immunology of Epstein-Barr virus infection

Epstein-Barr virus is a classic example of a persistent human virus that has caught the imagination of immunologists, virologists and oncologists because of the juxtaposition of a number of important properties. First, the ability of the virus to immortalize B lymphocytes in vitro has provided an antigen presenting cell in which all the latent antigens of the virus are displayed and are available for systematic study. Second, the virus presents an ideal system for studying the immune parameters that maintain latency and the consequences of disturbing this cell-virus relationship. Third, this wealth of immunological background has provided a platform for elucidating the role of the immune system in protection from viral-associated malignancies of B cell and epithelial cell origin. Finally, attention is now being directed towards the development of vaccine formulations which might have broad application in the control of human malignancies.     

Differential antigen requirements for protection against systemic and intranasal vaccinia virus challenges in mice

The development of a subunit vaccine for smallpox represents a potential strategy to avoid the safety concerns associated with replication-competent vaccinia virus. Preclinical studies to date with subunit smallpox vaccine candidates, however, have been limited by incomplete information regarding protective antigens and the requirement for multiple boost immunizations to afford protective immunity. Here we explore the protective efficacy of replication-incompetent, recombinant adenovirus serotype 35 (rAd35) vectors expressing the vaccinia virus intracellular mature virion (IMV) antigens A27L and L1R and extracellular enveloped virion (EEV) antigens A33R and B5R in a murine vaccinia virus challenge model. A single immunization with the rAd35-L1R vector effectively protected mice against a lethal systemic vaccinia virus challenge. The rAd35-L1R vector also proved more efficacious than the combination of four rAd35 vectors expressing A27L, L1R, A33R, and B5R. Moreover, serum containing L1R-specific neutralizing antibodies afforded postexposure prophylaxis after systemic vaccinia virus infection. In contrast, the combination of rAd35-L1R and rAd35-B5R vectors was required to protect mice against a lethal intranasal vaccinia virus challenge, suggesting that both IMV- and EEV-specific immune responses are important following intranasal infection. Taken together, these data demonstrate that different protective antigens are required based on the route of vaccinia virus challenge. These studies also suggest that rAd vectors warrant further assessment as candidate subunit smallpox vaccines.     

Non-pertussis components of combination vaccines: problems with potency testing.

Vaccines comprising combinations of diphtheria, tetanus and pertussis (DTP) with Haemophilus influenzae type b polysaccharide-protein conjugate (Hib), inactivated poliomyelitis virus (IPV) and hepatitis B virus (HBV) are already available, and new combinations using acellular pertussis components in a triple vaccine (DTaP) are under development. Evidence to date has shown that control of the efficacy, safety and stability of combination vaccines cannot be based on information already available on the individual components or existing licensed formulations. Several examples of immunological interference between components of a combination vaccine have been observed both in clinical trials and in laboratory tests. Examples of these for D, T and Hib components in DTP and DTaP combinations have been investigated.     

Biotechnology and vaccines: application of functional genomics to Neisseria meningitidis and other bacterial pathogens

Since its introduction, vaccinology has been very effective in preventing infectious diseases. However, in several cases, the conventional approach to identify protective antigens, based on biochemical, immunological and microbiological methods, has failed to deliver successful vaccine candidates against major bacterial pathogens. The recent development of powerful biotechnological tools applied to genome-based approaches has revolutionized vaccine development, biological research and clinical diagnostics. The availability of a genome provides an inclusive virtual catalogue of all the potential antigens from which it is possible to select the molecules that are likely to be more effective. Here, we describe the use of "reverse vaccinology", which has been successful in the identification of potential vaccines candidates against Neisseria meningitidis serogroup B and review the use of functional genomics approaches as DNA microarrays, proteomics and comparative genome analysis for the identification of virulence factors and novel vaccine candidates. In addition, we describe the potential of these powerful technologies in understanding the pathogenesis of various bacteria.     

Bacterial otitis media: current vaccine development strategies

Otitis media is the most common reason for children less than 5 years of age to visit a medical practitioner. Whilst the disease rarely results in death, there is significant associated morbidity. The most common complication is loss of hearing at a critical stage of the development of speech, language and cognitive abilities in children. The cause and pathogenesis of otitis media is multifactorial. Among the contributing factors, the single most important are viral and bacterial infections. Infection with respiratory syncytial virus, influenza viruses, para-influenza viruses, enteroviruses and adenovirus are most commonly associated with acute and chronic otitis media. Streptococcus pneumoniae, non-typeable Haemophilus influenzae and Moraxella catarrhalis are the most commonly isolated bacteria from the middle ears of children with otitis media. Treatment of otitis media has largely relied on the administration of antimicrobials and surgical intervention. However, attention has recently focused on the development of a vaccine. For a vaccine to be effective against bacterial otitis media, it must, at the very least, contain antigens that induce a protective immune response in the middle ear against the three most common infecting bacteria. Whilst over the past decade there has been significant progress in the development of vaccines against invasive S. pneumoniae disease, these vaccines are less efficacious for otitis media. The search for candidate vaccine antigens for non-typeable H. influenzae are well advanced whilst less progress has been made for M. catarrhalis. No human studies have been conducted for non-typeable H. influenzae or M. catarrhalis and the concept of a tribacterial vaccine remains to be tested in animal models. Only when vaccine antigens are determined and an understanding of the immune responses induced in the middle ear by infection and immunization is gained will the formulation of a tribacterial vaccine against otitis media be possible.     

Schistosoma spp.: progress toward a defined vaccine

During the last 2 decades, much was learned concerning the nature of acquired immunity in schistosomiasis, under experimental as well as field conditions. The knowledge is being applied now to design of a defined vaccine against this major parasitic disease. Several Schistosoma spp. antigens have been purified and shown to potentially protect in vivo. Determination of a method for presenting these antigens that will induce an optimal combination of cellular and humoral immune responses remains a critical step in vaccine development.     

A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice

It has been shown previously that measles virus (MV) can be successfully used to express foreign proteins (M. Singh and M. A. Billeter, J. Gen. Virol. 80:101-106, 1998). To develop an inexpensive MV-based vaccine, we generated recombinant MVs that produce structural proteins of hepatitis B virus (HBV). A recombinant virus that expressed the HBV small surface antigen (HBsAg) was analyzed in terms of its replication characteristics, its genetic stability in cell culture, and its immunogenic potential in genetically modified mice. Although this virus showed a progression of replication slightly slower than that of the parental MV, it appeared to stably maintain the added genetic information; it uniformly expressed the appropriately glycosylated HBsAg after 10 serial passages. Genetically modified mice inoculated with this recombinant MV produced humoral immune responses against both HBsAg and MV proteins.     

Mycobacterial proteins--immune targets for antituberculous subunit vaccine

Cellular and humoral immunity induced by Mycobacterium tuberculosis has led to identification of newer vaccine candidates, but despite this, many questions concerning the protection against tuberculosis remain unanswered. Recent progress in this field has centered on T cell subset responses and cytokines that these cells secrete. There has been a steady progress in identification and characterization of several classes of major mycobacterial proteins which includes secretory/export proteins, cell wall associated proteins, heat shock proteins and cytoplasmic proteins. The protein antigens are now believed to represent the key protective immunity inducing antigens in the bacillus. In this review, various mycobacterial protein antigens of vaccination potential are compared for their efficacy in light of current immunological knowledge.     

Characterization of hepatitis B virus DNA polymerase

Hepatitis B virus (HBV) particles were separated from the blood-plasma containing HBe and HBs antigens (subtype adr) and the nature of the endogenous DNA polymerase in the HBV core particles was studied. The HBV endogenous DNA polymerase activity was examined under the conditions used for preparation of HBV vaccine. The endogenous DNA polymerase activity was reduced slowly upon the heat treatment or the formalin treatment. The reductions of the activity were 65% and 70% upon the heat treatment at 60 C for 10 hr and the formalin treatment at 37 C for 90 hr, respectively. Properties of the HBV endogenous DNA polymerase were studied by utilizing specific inhibitors against the eukaryotic DNA polymerases. Our results showed that the HBV endogenous DNA polymerase is resistant to aphidicolin and N-ethylmaleimide, and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, phosphonoformic acid and 9-beta-D-arabinofuranosyladenosine 5'-triphosphate.     

Optimisation of prime-boost immunization in mice using novel protein-based and recombinant vaccinia (tiantan)-based HBV vaccine

Background

A therapeutic vaccine for chronic hepatitis B virus (HBV) infection that enhances virus-specific cellular immune responses is urgently needed. The “prime–boost” regimen is a widely used vaccine strategy against many persistence infections. However, few reports have addressed this strategy applying for HBV therapeutic vaccine development.

Methodology/Principal Findings

To develop an effective HBV therapeutic vaccine, we constructed a recombinant vaccinia virus (Tiantan) containing the S+PreS1 fusion antigen (RVJSS1) combined with the HBV particle-like subunit vaccine HBVSS1 to explore the most effective prime–boost regimen against HBV. The immune responses to different prime–boost regimens were assessed in C57BL/C mice by ELISA, ELISpot assay and Intracellular cytokine staining analysis. Among the combinations tested, an HBV protein particle vaccine priming and recombinant vaccinia virus boosting strategy accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres as well as the strongest multi-antigen (PreS1, and S)-specific cellular immune response. HBSS1 protein prime/RVJSS1 boost immunization was also generated more significant level of both CD4+ and CD8+ T cell responses for Th1 cytokines (TNF-α and IFN-γ).

Conclusions

The HBSS1 protein-vaccine prime plus RVJSS1 vector boost elicits specific antibody as well as CD4 and CD8 cells secreting Th1-like cytokines, and these immune responses may be important parameters for the future HBV therapeutic vaccines.     

A Chimeric Dengue Virus Vaccine using Japanese Encephalitis Virus Vaccine Strain SA14-14-2 as Backbone Is Immunogenic and Protective against Either Parental Virus in Mice and Nonhuman Primates

The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.