137 Interplay between host defenses and viral anti-defenses as a major factor of viral cytopathogenicity

The prevailing paradigm posits that virus-induced cellular injuries (cytopathic effect, CPE) are caused by hijacking of cellular substrates, energy, and infrastructure by the pathogens for the needs of their reproduction. However, this appears to be not the sole, and even not the most important, mechanism of cellular pathology triggered by viral infections. There is ground to believe that the most severe harm may come not from viral reproduction as such but rather from (miscalculated) host defenses as well as from viral anti-defensive activities. The experiments to be presented strongly support this notion. By using as a model system HeLa cells infected with mengovirus (a strain of encephalomyocarditis virus, a lytic picornavirus), we show that the major signs of CPE caused by this virus can be uncoupled from its reproduction. This can be achieved by partial mutual disarmament of the virus (by mutational inactivation of one of its anti-defensive “security” proteins, the leader protein) and the host (by chemical inhibition of one of its defensive innate immunity mechanisms, apoptosis). Under such conditions, the appearance of major cellular injuries is postponed until well after the completion of the viral reproduction. Remarkably, a more profound disarmament of the virus (by additional deletion of its second security protein, 2A) accompanied with a marked suppression of the viral reproduction leads to a faster death of the infected apoptosis-deficient cells due primarily to their defensive suicidal programmed necrosis. Thus, efficient strategies to ameliorate virus-induced injuries may include measures aimed at suppression of not only viral reproduction or viral anti-host functions but also of host defenses.

His main scientific interests are focused on diverse aspects of molecular and cellular biology of RNA-containing viruses (primarily picornaviruses) such as non-genetic and genetic interactions (co-discovery of complementation between non-enveloped RNA viruses; first biochemical proof of the intermolecular recombination between RNA genomes; demonstration of the existence of the non-replicative mechanism of this process), synthesis of viral proteins (first cell-free system for the faithful translation of picornavirus proteins; structural and functional analysis of translational cis-acting element of picornaviral RNAs; discovery and characterization of non-canonical translation initiation factors required for the synthesis of picornaviral proteins; identification of a protease involved in processing of the viral polyprotein; discovery of picornaviral leader proteins; first translational mapping of the flavivirus genome), replication of viral RNA (first evidence for involvement of host proteins in the synthesis of picornavirus RNA; characterization of viral 2C protein involved in the viral replication; functional and structural characterization of replicative cis-elements of the picornavirus genomes), neurovirulence and attenuation of picornaviruses (first mapping of viral phenotypic traits by recombinational analysis; discovery of dependence of attenuation on modulation of viral translation), cellular response to picornaviral infection (discovery of the ability of picornaviruses to trigger and suppress apoptotic response of the cells; discovery of the ability of picornaviruses to permeabilize the nuclear envelope by different mechanisms; demonstration that major picornavirus-induced cellular damages may be caused by interplay between host defense and viral anti-defenses and may be uncoupled from viral reproduction; proposal on the existence of a distinct set of viral proteins, dubbed security proteins, specifically dedicated to the anti-defensive activities), evolution (roles of changes in attenuation determinants, antigenic properties and recombination in the evolution of vaccine polioviruses; molecular epidemiology of poliomyelitis). He actively participated in the world struggle against polio. He is the author of over 175 papers on different aspects of virology, HIV/ AIDS and vaccine development. Esparza has served in numerous national and international scientific advisory committees and boards, receiving numerous awards in recognition to his many contributions to global health. He is a member of the National Academy of Medicine of Venezuela and of the Royal Academy of Doctors of Spain.
He received his MD degree in his native country of Venezuela (1968) and a PhD in Virology and Cell Biology from Baylor College of Medicine in Houston, TX (1974).
He currently lives with his wife in Seattle, WA and has a daughter and grandson.  One of the priorities for fundamental biomedical science is the discovery of the molecular-genetic basis of HIV pathogenesis. The progress in this area widens the possibilities for further improvement of HIV/AIDS prevention and therapy approaches. In this framework we have studied the population distribution of CCR5 rs333, CCR2 rs1799864, SDF1 rs1801157, HCP5 rs2395029, HLA-C rs9264942 and HLA-B*5701 genetic polymorphisms associated with susceptibility to HIV and AIDS and antiviral drugs intolerance among 1120 seronegative individuals comprising 12 ethnically distinct groups inhabiting Russia, Belarus, Ukraine, Kazakhstan, Kyrgyzstan and Moldova(Pomors, Russians(Vologda and Pskov region), Belarus, Ukrainians, Gagauz, Udmurts, Tatars, Chechens, Kazakhs, Kyrgyz, Tuvinians). The rs333 variant is represented at most in the population of Pomors. The homozygotes who have significantly lower risk of HIV infection comprising 3% of the population. The least frequency of CCR5 polymorphism was described for Chechens, Kazakhs, Kyrgyz, Tuvinians: 6, 6, 5 and 2% correspondingly. The highest incidence of CCR2 polymorphism was described for Chechens, Kazakhs and Kyrgyz. The other alleles under study did not reveal significant differences in distribution. The protective effect of the studied polymorphisms in CCR5, CCR2, и SDF1 genes is characterized as cumulative. Based on the frequencies of three-loci genotypes we have established the values of relative hazard of AIDS onset and relative hazard of AIDS-caused death for the populations under study. The risks fall in range 0.79-0.94 and 0.76-0.93, correspondingly. We have found that interpopulation genetic variability confers for statistically relevant differences in the hazards estimated. HIV-1 infects CD4+ T-lymphocyte by cell free viral particles released from the surface of effector cells or after the formation of intercellular contact between infected cell and healthy lymphocyte. The last way of infection was designated as cell-to-cell viral transmission. The current studies suggest that cell-tocell transmission of HIV occurs in peripheral lymph nodes at the early stage of infection and is more efficient pathway of virus dissemination than cell-free infection. One of the most accepted models of HIV transmission is the formation of virological synapse (VS); tight intercellular adhesion junction sharing structural similarity with immunological synapse. VS forms after HIV surface protein gp120 expressed on the plasma membrane of infected cells in prefusogenic conformation recognizes CD4 receptor on target cell. Viruses transmitted across VS undergo endocytosis by target cells, so that viral entry occurs via endocytic compartments and not via cell surface. This feature of cell-to-cell transmission accounts for the resistance of HIV to the neutralizing antibodies. Although the different stages of HIV cell-to-cell transmission have been extensively studied, the levels of correlation between virus transmission and targets infection were not accurately quantified. To fill up the gap in our understanding of cell-mediated viral infection, we have developed a new methods and improved vectors to quantify the levels of VS formation, cell-to-cell infection, and macromolecular transport across the cell-cell contact. New T cell lines stably producing recombinant virus like particles and reporter RNA, as well as modified target cells were generated. Development of an effective and safe HIV vaccine presents unique challenges. Despite the progress in understanding the biology of HIV-1 and its recognition by the human immune system, we have not yet developed an efficacious HIV-1 vaccine. During the last years, the results were obtained that has given hope that vaccination may prevent HIV-1 acquisition. The results of post-RV144 correlates and sieve analyses suggest that vaccine worked probably involving an immunological mechanism in the second variable loop (V2). A high resolution structure of the envelope trimer was solved, and this information is being used for the design of new generations of vaccines aimed at inducing protective antibodies. New techniques have facilitated the discovery of new human broadly neutralizing antibodies (BrNAbs) that target and delineate diverse conserved epitopes on the envelope glycoprotein spike. The epitopes of these BrNAbs can serve as templates for immunogen design aimed to induce similar antibodies. A separate goal of HIV vaccine research is to identify the best strategy for immunization and delivery agents. Several vectors and adjuvants have been developed. Significant advances have been made towards the development of tools to assess preclinical efficacy and understanding the correlates of protection in non human primate models. We observe the preventive potential of microbicides, PrEP, and earlier antiretroviral treatment of infected individuals has been demonstrated. All of these interventions may have a significant impact in reducing the incidence of HIV infections. However a safe and efficacious vaccine will be the most efficient way to control global HIV-1 pandemic, and scientific opportunities look promising. The measurement of immune responses directed specifically against the HIV is critical for understanding the interplay between the virus and the host immune system. By characterizing the immunological correlates of protection against HIV infection, such measurements will aid in the development of efficacious prophylactic vaccine. To improve vaccine antigens and adjuvant, it is also necessary to asses a similarity of vaccine -and virus -induced immune responses The evaluation of antigenspecific humoral response includes measurement amount and specificity of vaccine-induced antibodies (in ELISA or WB), their neutralizing activity and ADCC or ADCVI. ELISPOT, intracellular cytokine flow cytometry assays and Luminex are the most common assays to determine CTL response. They all determine immune response by the detection of the cytokines (IFNgamma, TNF-alpha, IL2) secreted by cells upon antigenstimulation. MHC tetramer binding assay measures the absolute number of cells that recognize a particular epitope without providing any information regarding the functionality of the cells. Two surface glycoproteins hemagglutinin and neuraminidase are the main antigens for obtaining traditional flu vaccines due to their high immunogenicity and ability to induce generation of neutralizing antibodies. High genetically instability of influenza virus provides antigenic diversity of these glycoproteins between strains. Subunit vaccines based on one of these surface antigens are characterized by narrow specificity. Modern vaccines should provide protection against several or all subtypes of influenza A viruses. In addition sporadic influenza outbreaks of newly emerged influenza A viruses may cause the next pandemic.

Abstracts
Today there are no flu vaccines of a wide range of action, so-called "universal" vaccines. Several flu vaccines with a broad cross-protectivity are tested in different phases of clinical trials.
In this report the analysis of the modern researches directed on creation of flu "universal" vaccines including data obtained by researchers of Gamaleya institute of epidemiology and microbiology is carried out.
Genetic immunization technology is used for development of "universal" vaccine. Recombinant pseudoadenoviral particles coding of influenza virus conservative antigens were used as a vector for gene delivery. Polypeptide, containing hemagglutinin conservative epitopes, was used in addition to traditional influenza conservative antigens M1, M2 and NP. Animal experiments demonstrated that developed vaccines were very efficient against different influenza subtypes (80%-100% survivability). year. Currently the only licensed anti-tuberculosis vaccine is Bacille Calmette-Guérin (BCG). This live attenuated vaccine has been in use for over 80 years and has displayed up to 80% efficacy against serious forms of the disease, e.g., meningitis, in children. However, efficacy in adults against pulmonary tuberculosis ranges from 0 to 80% in different trials. The protective immunity generated by BCG decreases with time, and almost disappears after 20-25 years, resulting in a vulnerability of the adult population to primary infection and reactivation of latent TB. Thus, development of an effective preventive vaccine and new "post-infection" vaccine are still important aims of vaccinology.
In this study we report the development of novel subunit protein-based vaccine against tuberculosis. Our vaccine consists of two recombinant mycobacterium proteins -Ag85A and fusion protein ESAT6-CFP10. These antigens are strongly recognized by T cells and they have demonstrated protective efficacy in animal models. However, because the protein component of the subunit vaccine is poorly immunogenic, the recombinant protein vaccine was adjuvanted with novel, three-component adjuvant system composed of the dextran, cationic DEAEdextran and oligodeoxynucleotide CpG. This adjuvant was chosen on the basis of its ability to induce strong protective immunity in animal models of M. tuberculosis infection, by delivering a TLR9 ligand into the endosomal pathway. Recombinant vaccine proteins possess dextranbinding domain, which helps create «depot» effect and provide prolonged interaction of vaccine antigen with T-cells.
We investigated immunogenicity and protection properties of our vaccine on mice and guinea pigs models of tuberculosis, and also performed toxicity studies. We demonstrated that our subunit vaccine stimulates th1 immune response and is protective against tuberculosis with the same efficacy as BCG. Furthermore, our vaccine showed good result in toxicity studies in mice, guinea pigs and rabbits.

Broadly neutralizing antibodies for serotherapy and vaccine design Antonio Lanzavecchia Institute for Research in Biomedicine, Bellinzona and Institute of Microbiology, DE
Memory B lymphocytes and long-lived plasma cells represent a repository of the antigenic experience of an individual. By analyzing the specificity and function of these cells we can gain insights into the human immune response to pathogens and vaccines, identify correlates of protection, and isolate neutralizing antibodies and protective T cells. To interrogate human memory B cell and plasma cell repertoires we developed two culture-based high-throughput methods that are used to isolate, with high efficiency, human monoclonal antibodies of distinctive specificities. Unusually potent neutralizing antibodies against human cytomegalovirus were isolated from infected donors and used to identify the viral ligands and to design an experimental vaccine. We also isolated antibodies of exceptional breadth, such as a pan-influenza A neutralizing antibody and an antibody that neutralizes both respiratory syncytial virus and metapneumovirus. By targeting conserved structures, these broadly neutralizing antibodies are less prone to select escape mutants and are therefore promising candidates for prophylaxis and therapy of infections as well as tools for the design of improved subunit vaccines. Forty percent of the world's population is at risk for dengue, and 100 million apparent infections are estimated to occur annually. In a minority of cases, dengue fever progresses to severe disease at the time of defervescence, inducing a capillary leak syndrome accompanied by thrombocytopenia that can result in shock and organ failure. Thus the medical need for a dengue vaccine is obvious. The four dengue virus serotypes are transmitted by Aedes mosquitoes, and protective immunity against the infecting serotype (homotypic immunity) is long lived and thought to be conferred by virus-neutralizing antibodies and some T cell help. However, antibodies induced by one serotype often cross-react with the other serotypes. If antibodies fail to neutralize and instead opsonize, they can enable the virus to infect IgG receptorbearing cells. This phenomenon, termed antibodydependent enhancement, is thought to play a role in the pathogenesis of severe dengue, and is of concern to vaccine developers. Several investigational dengue vaccines are currently in clinical development, and many different vaccination approaches are being explored. These span from live-attenuated dengue viruses to chimera between yellow fever and dengue viruses, to subunit, DNA, and whole virus inactivated vaccines. A recent Phase 2b study of the vaccine most advanced in development did not show statistically significant protection against disease due to any serotype, adding much uncertainty to the entire field. Our understanding of the contributions of humoral and cell mediated responses to protection and to pathogenesis is clearly less than perfect, and selection, presentation and adjuvantation of antigens need thought and attention. EV71-induced HFMD is usually characterized by the formation of maculopapular or vesicular lesions on the skin and oral mucosa, especially on the palms, soles, and mouth.. There were a greater number of fatal cases with brainstem encephalitis, pulmonary edema and/or hemorrhage, and cardiopulmonary collapse.

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Developing effective vaccines is considered a top choice among all control measures. We evaluated the ability of inactivated virus vaccine to elicit neutralizing antibody and to provide protection against lethal infection of EV71 in suckling mice. The purity of EV71 vaccine was up to 96.8% by HPLC identification. The purified EV71 vaccine induced high levels of neutralizing antibodies, these antibodies were shown to be protective against lethal infection when passively transferred to susceptible newborn mice. With a challenge dose of 50LD50 virus/ mouse, suckling mice born to dams immunized with inactivated virus showed 100% survival. In preliminary animal trial, no side effects were detected when monkeys were immunized with purified EV71 vaccine either at normal or large doses.
The vaccine was approved of the clinical evaluation in 2009. The phase 1/2/3 clinical trail was completed in March, 2013. The data suggested that the inactivated EV71 vaccine had a clinically acceptable safety profile and good immunogenicity in healthy children and infants. Our data indicated that inactivated EV71 vaccine is the choice of vaccine preparation capable of fulfilling the demand for effective control.

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The leading etiological agents of viral gastroenteritis are group A rotaviruses, noroviruses, adenoviruses (type 40, 41) and astroviruses in China in the last 20 years. The prevailing serotypes of rotaviruses are G type 1, 2, 3, 4 and 9, while G8 has been emerged recently in China.

VLPs and particle vaccines
Luigi Buonaguro, MD Ist. Naz.Tumori "Fond. G. Pascale," IT VLPs represent a powerful tool for vaccine development, representing the closest strategy to the native viruses for displaying and delivering conformational epitopes, with improved induction of antibodies and immune response in its whole. Moreover, the lack of genetic material makes VLPs the "safer counterpart" of live attenuated or killed viral vaccines which, indeed, may induce limited but possible undesired effects.
VLP-based vaccines have been developed from both enveloped and non-enveloped viruses to target infectious and non-infectious diseases and are in various stages of development spanning preclinical evaluation to market. In particular, the only two preventive cancer vaccines licensed are based on VLP approach (HBV and HPV vaccines).
Overall, pre-clinical and clinical trials with different VLP-based vaccines have shown they are well tolerated and can be administered by a number of routes, including intramuscular, subcutaneous, oral or intranasal. VLP vaccines have also been demonstrated to be highly immunogenic and capable of stimulating protective immunity in a number of instances when administered with or without adjuvants. Their potent immunogenicity is a result of several factors, including the ability to incorporate key immunogenic properties of viruses into a single entity and to hit the cells relevant for initiate an effective adaptive immune response. We have previously reported the protective efficacy of Ad26/MVA vector regimens expressing SIVsmE543 Env/Gag/Pol antigens against SIVmac251 challenge.

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Here we assess the potential utility of adding a protein trimer boost to adenovirus based vaccine regimens. We produced stable SIV and HIV Env gp140 trimers and assessed the immunogenicity and protective efficacy of Ad prime, Env gp140 trimer boost regimens against SIVmac251 and SHIV-SF162P3 challenges in rhesus monkeys. Vector prime, protein boost vaccine regimens induced higher titer binding and neutralizing antibodies compared with protein only regimens. Clinical studies to evaluate Ad26/MVA and Ad26/ protein vaccine regimens are planned.

Strategies and mechanisms for vaccine induction of mucosal T cell immunity Jay A. Berzofsky Vaccine Branch, Center for Cancer Research, National Cancer Institute, NIH, US
Most HIV transmission is through mucosal surfaces. We previously found in mice that CD8 T cells must be in the local mucosa to prevent mucosal viral transmission. Intrarectal delivery was most effective, but not so practical. We devised a strategy to mimic intrarectal delivery through the oral route by encapsulating antigens in nanoparticles enclosed in microparticles that protected against stomach acid and enzymes and released the nanoparticles in the large intestine for update by dendritic cells. Different formulations resulted in selective release in the small or large intestine and T cell immunity in those sites, revealing a heretofore unknown subcompartmentalization between the small and large intestinal mucosal immune system. Delivery to the colon mimicked intrarectal delivery for protection of mice against intrarectal vaccinia virus challenge. We translated this to macaques, showing similar selective delivery and some preliminary evidence of efficacy in a SHIV intrarectal challenge model. The subcompartmentalization also implies that the homing receptors for T cells to traffic to the small and large intestine must differ. We found that indeed, small intestinal DCs induce T cells to home to the small intestine, whereas colonic DCs induce homing to the colon. This difference appears to correlate with different levels of retinoic acid production and differential induction of homing integrins and chemokine receptors. Besides the ability to mimic the benchmark intrarectal immunization by a more acceptable route, such subcompartmentalization may be useful for selective immunization, and understanding the mechanisms involved in homing may allow selective targeting of T cells to the right compartment. The second paradigm aimed at inducing CD8+ T-cell responses, and it led to the development of DNA vaccines and of live-recombinant viral vectors, especially poxviruses and adenoviruses (Ad). The concept was tested in the STEP and Phambili trials, using an Ad5 vector. The trials were stopped in 2007, after an interim review of STEP showed that the vaccine failed to prevent HIV infection or to decrease virus load in vaccinated volunteers who became infected, and even enhanced HIV acquisition in a subpopulation of vaccinated individuals.
The current wave of vaccine development is attempting to induce more complex immune responses and exploring novel approaches. The RV 144 trial in Thailand, which assessed the protective efficacy of a prime-boost protocol using a canarypox vectors followed by gp120 boosts, showed 31.2% efficacy in preventing HIV acquisition and presumptive immune correlates have been identified. The field is now exploring new leads that include the rational design of novel immunogens based on epitopes recognized by broadly neutralizing antibodies, live replicationcompetent vectors and the role of potentially protective non-neutralizing antibodies. The envelope gp120 of HIV contains an unusual high amount of N-glycans that serves as a dense shield to hide immunogenic epitopes on gp120 against the immune system. There exists a wide variety of glycan-binding agents with different specificities to recognize carbohydrate moieties present on the HIV envelope. Several of the carbohydrate-binding agents (CBA) are potent inhibitors of at least four different pathways of HIV to infect virus-susceptible cells and to be transmitted to virus-exposed individuals. These CBAs have been demonstrated (i) to inhibit cell-free virus infection of CD4+ T-lymphocytes and macrophages, (ii) to block syncytia formation between persistently HIV-infected (gp120-expressing) cells and uninfected cells, (iii) to prevent virus capture by DC-SIGN-expressing cells and (iv) to block transmission of DC-SIGN-captured virus particles to uninfected CD4+ T-lymphocytes. CBAs represent the only agents reported to be able to concomitantly interact with these four processes, which can be advantageous from a microbicide perspective. Interestingly, CBA drug pressure in cell culture forces the virus to select for mutant variants that have multiple N-glycan deletions in their envelope, and thus, to uncover immunogenic epitopes on gp120. Accumulation of such envelope mutations compromises the infectivity of the virus. Several gp120 antibodies show a markedly increased activity against such mutant viruses. CBAs may, therefore, represent an entirely novel therapeutic concept in that they inhibit virus entry into its susceptible cells, and prevent DC-SIGN-directed virus transmission, but in addition, they may trigger the host immune system as soon as drug-related (N-glycan) mutations appear in the viral envelope. The advent of triple drug highly active antiretroviral therapy in 1996 was a turning point in the battle to control HIV disease. For the first time, HIV replication could be effectively limited resulting in marked improvements in morbidity and mortality from HIV infection. However, the treatment regimens were complex with patients having to take many pills at different times during the day, and the regimens were associated with significant long term side effects. Subsequent treatment strategies have focused on simplification of dosing to facilitate adherence and better tolerated regimens. Various combination pills were developed beginning with Combivir ( zidovudine/ lamivudine) given twice daily and then Truvada (tenofovir DF/emtricitabine) given once daily in combination with a third or fourth drug. This led to the development of single tablet regimens (STR), a single pill that provided complete therapy for treatment of HIV infection. The first STR was Trizivir (zidovudine/ lamivudine/abacavir) given twice daily, which was followed by the once daily STRs Atripla ( tenofovir DF/emtricitabine/efavirenz), Complera (tenofovir DF/emtricitabine/rilpivirine), and Stribild (tenofovir DF, emtricitabine, cobicistat, elvitegravir), which are now the most commonly used medications for the initial treatment of HIV infection. New drugs are in development, including the tenofovir prodrug TAF (tenofovir alafenamide fumarate) and the integrase inhibitor dolutegravir, which may offer advantages in safety, efficacy, and simplicity of dosing, and could be included in future STRs. The mechanisms of male-to-female HIV-1 transmission mediated by semen are not well-understood. To decipher these mechanisms, we developed a system of cervico-vaginal tissue ex vivo to which HIV-1 is transmitted from semen under controlled laboratory conditions. Semen samples obtained from HIV-1infected and control men were analyzed for the presence of 21 cytokines, and HIV-1 transmission was simulated ex vivo by deposition on cervico-vaginal explants of virus suspended in semen or in PBS enriched with particular cytokines. In HIV-1-infected men, the cytokine spectrum was significantly changed, resulting in the establishment of new correlations and the strengthening of pre-existing correlations between different cytokines: HIV-1 infection increases the number of such correlations from 21 to 72. These changes in semen were local and different from ones in blood from the same individuals. One of the most upregulated seminal cytokines was IL-7, which enhanced replication of HIV-1 in cervico-vaginal tissue. This enhancement was associated with the suppression of apoptosis as evaluated from the expression of apoptotic markers, a decrease in the depletion of infected cells as evaluated from flow cytometry, and an increase in cell cycling as evaluated from Ki67 staining.

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In conclusion, HIV-1 establishes new strong correlations between various cytokines, thus imposing a high rigidity on their network that may contribute to the impaired capacity of the immune system to respond to microbial challenges. Seminal cytokines, in particular IL-7, may be key determinants of HIV-1 transmission from men to their uninfected female partners through vaginal intercourse and may become a new target for preventive and therapeutic strategies.

Alain Lafeuillade General Hospital, Toulon, FR
Combined antiretroviral therapy (cART) has dramatically increased life expectancy during HIV infection but must be taken life-long. Consequently, it carries problems of compliance, resistance, toxicity and cost. Finding a cure against HIV would resolve these issues. In theory, it could be either a "functional" or a "sterilizing" cure, but its main goal would be to keep HIV replication at bay without continuing cART. A couple of situations already exist where this has been achieved: -The Berlin patient who is free of cART more than 5 years following an allogenic bone marrow transplant from a donor with the delta 32 mutation on the CCR5 gene; -The "Mississippi child" who received cART within 31 hours after birth; -The "VISCONTI" patients who received cART at acute HIV infection and remained aviremic when this therapy was stopped a few years later; -Two patients who received allogenic stem cell transplantation for lymphomas and have no trace of HIV, although cART is still continued.
Developing therapeutic strategies toward a HIV cure needs a deep understanding of the mechanisms that allow HIV persistence in reservoirs despite cART. Using current knowledge, approaches trying to purge the HIV reservoir have reached clinical trials using vorinostat. These trials showed the need of more potent activators combined with an immunologic intervention to eliminate cells where latent HIV has been activated. However, as the HIV reservoir is probably more complex than initially thought, others strategies than reactivation have to be pursued, including gene therapy. The introduction of potent combination therapies in the mid-90s had a tremendous impact on AIDS mortality. However, drug resistance has been a major factor contributing to antiretroviral therapy failure. At present, reverse transcriptase (RT) inhibitors constitute the backbone of successful antiretroviral therapies. The HIV-1 RT is a heterodimer composed of subunits of 560 and 440 amino acids. Mutations in the RT-coding region selected during treatment with nucleoside RT inhibitors (NRTIs) confer resistance by altering discrimination between NRTIs and natural substrates (dNTPs), or by conferring a phosphorolytic activity (dependent on pyrophosphate or ATP) that unblocks the chainterminating inhibitor from the 3´ end of the DNA that is being synthesized. The most relevant mutations conferring resistance to RT inhibitors map within the DNA polymerase domain of the RT (first 260 residues), and this region is subjected to genotypic analysis in order to select the proper antiretroviral treatment.

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Despite the reasonable knowledge of the correlates between HIV genotype and the virological response to current therapies, our knowledge is still incomplete. The effects of antagonistic mutations and amino acid substitutions outside the DNA polymerase domain of the RT have been poorly characterized and examples will be given to illustrate the complexities of mutational patterns involved in resistance. In addition, I will provide examples of epistatic effects of HIV-1 protease and RT mutations that could affect viral fitness. Finally, an overview of mutational pathways and mechanisms of resistance to novel antiretroviral drugs (e.g. raltegravir, elvitegravir, etravirine, rilpivirine and maraviroc) will be briefly presented.  Current antivirals cannot efficiently control but not eliminate hepatitis B virus (HBV) in the 300 million carriers at risk to develop liver diseases and cancer, because HBV establishes a stable nuclear cccDNA in infected hepatocytes. Interferon can clear HBV but therapeutic dosing is limited by side effects. We found a mechanism by which nuclear viral DNA can specifically be degraded. In HBV-infected cells and primary human hepatocytes, human liver-needle biopsies and in vivo mouse models, interferon and even at much lower doses lymphotoxin--receptor activation up-regulated nuclear APOBEC3 family deaminases. Cytidine-deamination resulted in apurinic/apyrimidinic site formation and finally cccDNA degradation. No deamination was detected in genomic DNA since HBV-core protein targeted the APOBEC3 protein to cccDNA. Inducing nuclear APOBEC3 deaminases e.g. by triggering the lymphotoxin--receptor pathway will be of high interest for the development of new therapeutics. The combination with existing antivirals shows great promise to eliminate the virus in chronic hepatitis B.

Polio Vaccines: The Past, Present and the Future Konstantin Chumakov FDA Office of Vaccines Research and Review, US
Inactivated Polio Vaccine (IPV) and live Oral Polio Vaccine (OPV) are among the most successful and widely used vaccines in the history of medicine. The history of their development and subsequent evolution in response to changing epidemiological and socioeconomic circumstances will be reviewed along with new efforts to develop a new generation of polio vaccines. OPV was responsible for virtual elimination of the disease, and its worldwide eradication may be achieved in few years. Paradoxically, this monumental accomplishment demands introduction of a new generation of vaccines specifically designed for the new polio-free world to ensure the lasting success of the eradication campaign. Both current vaccines have their strengths and weaknesses: OPV is inexpensive and easy to administer, but its virus can revert to virulence and cause paralytic disease. IPV is highly efficient and safe but is more expensive, requires several intramuscular injections, and does not induce fully adequate mucosal immunity. New vaccines must combine the best characteristics of both. Lower cost of IPV and broader immune response could be achieved by using adjuvants, alternative routes of administration, and increasing production yield. Production of IPV is associated with biosecurity concerns because it is made from highly virulent strains. Novel approaches to reduce virulence of strains used for IPV manufacture will be discussed. Development of a more genetically stable OPV is underway by attempting to reduce rates of mutations and recombination. Polio vaccines were a paradigm for many other vaccines, and lessons learned from their evolution could help develop other prophylactic products. While the pathogenesis of ATLL is incompletely understood, the viral regulatory and oncoprotein Tax is considered to play a central role. To better understand the role of Tax and to develop animal models of disease we have generated transgenic mouse models which have remarkably reproduced the clinical, pathological, hematological and molecular features of human disease. Studies on the latter have highlighted the important role of chemokines in the infiltrative and migratory properties of malignant cells, allowed the identification of specific cellular signalling pathways involved and the employment of specific inhibitors as treatment modalities. Treatment responses however have been limited and this can be attributed to the presence of cancer stem cells (CLCs). We will describe the identification and characteristics of the CLCs and demonstrate their important role in disease progression. In addition we will summarise studies employing proteomic approaches to identify unique CLC protein expression profiles which may serve as important targets towards eradicating the CLC population and which might serve as key therapeutics.

Replication and assembly of filoviruses Larissa Kolesnikova, Gordian Schudt, Olga Dolnik, and Stephan Becker Institut für Virologie, Philipps-Universität Marburg, DE
The family of Filoviridae comprises Marburg and Ebola virus which both cause severe life-threatening diseases characterized by high fever, rash, thrombocytopenia and hemorrhagic diathesis. The pathogenesis of the syndrome is not completely understood; probably the dynamic replication of filoviruses in the infected host leads to an uncoordinated immune response. Detailed understanding of the basic mechanisms of filoviral assembly and interaction with host cells is key to identify targets of antiviral intervention. The first sign of filovirus replication that can be detected microscopically in the infected cell is the formation of inclusions in the perinuclear region. Inclusions contain all filoviral nucleocapsid proteins (NP, VP35, VP30, VP24, and L) but also the matrix protein VP40 and a number of cellular proteins. Viral nucleocapsids are formed within the inclusions by specific interactions among the viral proteins. Mature nucleocapsids are transported across the cytoplasm to the plasma membrane with the help of the actin cytoskeleton. In the cell periphery nucleocapsids are associated with the matrix protein and channeled into filopodia, the site of filoviral release. Nucleocapsids inside filopodia are cotransported together with the unconventional motor protein myosin 10. Transport of nucleocapsids and release of viral particles is supported by the cellular ESCRT machinery. An intrinsic property of all retroviruses is their capacity to establish a state of latent infection in addition to active viral replication. Both genetic and epigenetic factors contribute to silence the integrated provirus and are nowadays targets of experimental approaches aiming at reactivating virus expression in order to kill the infected cells either by immune-mediated mechanisms (such as CTL) or by pharmacological agents. We have recently uncovered an unsuspected pathway triggered by essential aminoacid starvation and involving the downregulation of HDAC4, a class II HDAC, leading to a significant uprising of latent HIV-1 infection in chronically infected cell lines, such as ACH-2 cells, expressing this enzyme, but not in similar cell lines, such as U1, negative for HDAC4 expression. Both pharmacological targeting of HDAC4 and siRNAmediated downregulation of its expression gave the same result. A similar phenomenon was observed for heterologous transgenes, driven by different promoters, introduced for gene therapy purposes, but not for their endogenous counterparts. This observation suggests that the mTOR-independent pathway triggered by aminoacid starvation and involving HDAC4 modulation represents or contributes to an intracellular response to foreign nucleic acids (I. Palmisano et al., PNASPlus 2012). More recent results with a broader variety of HIV-1 latency models will be discussed. Proteins, particularly viral proteins, can be multifunctional, but the mechanism(s) behind this trait are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry and cellular microscopy that the Ebola virus VP40 protein rearranges into different structures, each with a distinct and essential function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer trafficks to the cellular membrane. There, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multi-layered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly, but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle, and demonstrate how a single, wild-type, unmodified polypeptide can assemble into different structures for different functions.
Schmallenberg virus (SBV) is a novel orthobunyavirus of ruminants that emerged in Europe in the summer of 2011. Although mild clinical signs are observed in infected adult animals, transplacental infection has been associated with the development of congenital malformations, stillbirths and abortions. SBV has spread dramatically across north-western Europe reaching 100% prevalence in some herds. Despite antibodies against SBV being found in a variety of animal species neither human cases nor positive serology in highly exposed humans have been detected. We have recently developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Specifically we developed a reverse genetics system that aided us in unraveling determinants of virulence.
Here, we report identification of a cellular factor that restricts SBV replication. While the human orthologue restricts SBV replication under different experimental conditions, no restriction is observed by the ovine counterparts. We show that the restriction is specific since SBV replication is restored under the presence of a known viral restriction factor antagonist. We also show that this viral restriction factor and SBV nucleocpasid co-localize at the cell membrane and in the perinuclear region and we partially unravel the mechanism of restriction. Based on these results we hypothesize that the lack of positive human serology to SBV is related to the ability of this cellular factor to restrict SBV replication and we are currently exploring if these findings can be extended to other bunyaviruses. CD4 T cell reconstitution in patients with HIV is associated with increased responses to conventional vaccines and improved resistance to opportunistic infections. However, prolonged therapy fails to replenish a capacity for immune control over HIV and patients who interrupt therapy inevitably rebound to pre-treatment viral burden in plasma. Only elite patients, termed NVS or natural virus suppressors (1), control viremia to undetectable levels in the absence of therapy. Our earlier studies on NVS patients showed that this group was distinguished from all other HIV patients by the presence of normal levels of CD56+ Vи2Vи2 T cells in blood (2) reflecting reconstitution of the T cell receptor repertoire through new cell synthesis (3). The CD56 marker identifies cytotoxic effector lymphocytes including NK, CD8,иии T and NKT subsets, but it's function remains unknown. We have now shown persistent depression of CD56 expression on CD8 T cells from HIV+ individuals except for NVS patients where the levels approach those found in normal controls. The CD56+ CD8 T cell subset is highly responsive to stimulus, expresses high levels of perforin/granzyme and the small population remaining in HIV patients on therapy, also express the immune exhaustion marker TIM-3, indicating cells may be lost through an apoptosis mechanism. The persisting defect in CD56 expression for two T cells subsets is consistent with a lack of cytolytic effector function and is present in patients with complete virus suppression for many years due to treatment, long after the T cell population is refreshed by new cell synthesis. The block to lytic effector function may explain why treated patients fail to eradicate viral reservoirs; this mechanism is a key target for new therapies designed to cure HIV disease.
HIV-1 Nef promotes viral replication by downmodulating a number of cell surface transmembrane proteins, such as CD4, MHC-I and MHC-II, which are targeted by Nef to various degradation pathways. Nef is also responsible for downregulation of cellular cholesterol transporter ABCA1, and this effect contributes to development of atherosclerosis in HIV infected patients. Surprisingly, in contrast to CD4 and MHC I, to which Nef has to bind to exert downregulation, binding to ABCA1 turned out to be unnecessary for inactivation of ABCA1 by Nef. Here, we identified a novel mechanism by which Nef influences activity of host cell and viral proteins. We show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which is essential for folding and maturation of glycosylated proteins. Nef disrupts calnexin interaction with ABCA1, thus impairing functionality of this protein, but increases affinity and enhances interaction of calnexin with gp160, promoting maturation and functionality of viral Env proteins. Knock-down of calnexin lead to reduced fusion activity of HIV-1 envelope and reduced virion infectivity, as well as to defective cholesterol efflux, which is mediated by ABCA1. However, gp160 and ABCA1 interacted with calnexin differently: while gp160 binding to calnexin was dependent on glycosylation, interaction of ABCA1 with calnexin was glycosylationindependent. Therefore, Nef binds to calnexin and stimulates interaction between calnexin and gp160 at the expense of ABCA1 and probably other ER proteins. These results provide a mechanistic explanation for previously unexplained effect of Nef on functionality of ABCA1, and suggest a mechanism for upregulation of HIV infectivity by Nef through stimulation of Env maturation. The prevailing paradigm posits that virus-induced cellular injuries (cytopathic effect, CPE) are caused by hijacking of cellular substrates, energy, and infrastructure by the pathogens for the needs of their reproduction. However, this appears to be not the sole, and even not the most important, mechanism of cellular pathology triggered by viral infections. There is ground to believe that the most severe harm may come not from viral reproduction as such but rather from (miscalculated) host defenses as well as from viral anti-defensive activities. The experiments to be presented strongly support this notion. By using as a model system HeLa cells infected with mengovirus (a strain of encephalomyocarditis virus, a lytic picornavirus), we show that the major signs of CPE caused by this virus can be uncoupled from its reproduction. This can be achieved by partial mutual disarmament of the virus (by mutational inactivation of one of its anti-defensive "security" proteins, the leader protein) and the host (by chemical inhibition of one of its defensive innate immunity mechanisms, apoptosis). Under such conditions, the appearance of major cellular injuries is postponed until well after the completion of the viral reproduction. Remarkably, a more profound disarmament of the virus (by additional deletion of its second security protein, 2A) accompanied with a marked suppression of the viral reproduction leads to a faster death of the infected apoptosis-deficient cells due primarily to their defensive suicidal programmed necrosis. Thus, efficient strategies to ameliorate virus-induced injuries may include measures aimed at suppression of not only viral reproduction or viral anti-host functions but also of host defenses Measles is the poster child for establishment of life long immunity to re-infection. This is associated with a prolonged persistence of measles virus (MeV) RNA in blood and lymphoid tissue. The live attenuated vaccine is also effective, but the immune responses are less robust and of somewhat shorter duration than after natural infection. Through study of a variety of experimental DNA and virus-vectored vaccines in rhesus macaques followed by wild type MeV challenge, we have identified 4 levels of protective immunity: 1) protection from both rash and viremia; 2) protection from rash, but not viremia; 3) protection from rash and viremia, but not infection; 4) no protection with possibility of enhanced disease. Protection from rash requires high levels of high avidity MeV neutralizing antibody, but this is not sufficient to protect from viremia. MeV-specific T cells alone do not protect from rash or viremia, but lead to more rapid clearance of MeV RNA from blood. Antibody and T cell responses to the hemagglutinin (H) and fusion (F) surface glycoproteins protect from rash and viremia, but not infection leading to late appearance of viral RNA in blood. Parenteral or deep lung respiratory delivery of the live virus vaccine protects against rash, viremia and infection. Further studies are needed to determine if experimental vaccines require additional antigens or induction of a different type of immune response to recapitulate the protection provided by the live attenuated vaccine virus and natural infection.

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Dengue vector control: critical needs and opportunities for helping to control the dengue pandemic Barry Beaty Department of Microbiology, Immunology, and Pathology, Colorado State University, US Aedes aegypti mosquito control is currently the only option for controlling and preventing epidemic dengue and dengue hemorrhagic fever. However, current approaches for vector control are not stemming the rising tide of dengue disease throughout the tropical world. In the absence of a vaccine, new and effective approaches are needed to improve vector control. Novel approaches to prevent dengue virus transmission will be described and discussed, including 1) the Casa Segura approach to prevent dengue transmission in the home, 2) development of a new generation of molecular mosquitocides to address the rise of resistance to existing insecticides, and 3) engineering dengue virus resistant mosquitoes. There is a public health imperative to increase the armamentarium for vector control. As genome wide association studies plus whole genome sequence analyses for complex human disease determinants are expanding, it seems useful to develop strategies to facilitate large data sharing, rapid replication and validation of provocative statistical associations that straddle the threshold for genome wide significance. At this conference, we shall announce GWATCH, (Genome Wide Association Tracks Chromosome Highway) a web based data release platform that can freely display and inspect unabridged genome tracked association data without compromising privacy or Informed Consent constrictions, allowing for rapid discovery and replication opportunities. We illustrate the utility with HIV-AIDS resistance genes screened in combined large multicenter cohort studies GWAS (MACS, HGDS, MHGS, ALLIVE , LSOCA HOMER) developed and studied over the last decades. At the present state of knowledge, Sochi virus seems to be the most dangerous representative of DOBV. Virus diagnostics in patients was accomplished by immunofluorescence assay, serotyping of neutralizing antibodies, and RT-PCR amplification of viral genome segments. In phylogenetic analyses we found a spatial clustering of the viral nucleotide sequences derived from patients and mice trapped at different localities of the Russian Black Sea coast region demonstrating Sochi virus as the causal pathogenic agent in humans. We currently oversee in detail the clinical courses of 51 patients with confirmed Sochi virus infection. The case fatality rate was determined to be as high as 14%. Nearly 60% of clinical courses were defined as severe (including deaths) and nearly 40% as moderate. Four times more males than females were affected. Quite unusual for hantavirus disease, also young people became ill due to Sochi virus infection; 10% of patients were found between 7 and 15 years old and the age average of all patients was not much higher than 30 years.

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There is an urgent need to monitor the epidemiology of the new virus -not only because of its healththreatening character in this particular geographical area but also because of its potential ability to overcome host species barriers. Colonization of nearly related host species, as A. flavicollis or A. sylvaticus, by the virus could dramatically increase its geographical spread and consequently further enhance the danger for the human population. The capacity of Rift Valley fever virus (RVFV) to spread into new territories by crossing significant natural geographic barriers, re-emerge in endemic regions after long periods of silence to cause large outbreaks in human and animal populations constitute a formidable challenge for public and veterinary health authorities as well as for scientific communities worldwide. In spite of recent advances in research on RVFV pathogenesis, molecular epidemiology, outbreak prediction, development of new diagnostic tools and vaccines, some fundamental aspects of the epidemiology and ecology of the virus remain elusive. Large outbreaks of RVF are associated with anomalous high rainfalls leading to massive flooding and the resultant swarms of competent mosquito vectors transmitting the virus to a wide range of susceptible vertebrate species. However, the exact mechanism of RVFV natural transmission during interepizootic periods remains largely unknown, including the postulated long-term virus persistence in transovarially infected eggs of floodwater Aedes mosquito species, and the role of wild mammals as reservoirs. The presence of competent mosquito vectors in countries free of RVF, the wide range of mammals susceptible to the virus, the global changes in climate, and increased animal trade and travel are some of the factors which might contribute to international spread. This presentation provides the background to the major outbreaks, molecular biology and epidemiology of RVFV, and overviews aspects of ecology, host and vector range which make the virus a potential global emerging threat.  2012). These cancers include all cervical cancers, the vast majority of anal cancers and approximately half of cancers of the vulva, vagina, and penis. In the head and neck, HPV is clearly involved in a fraction of cancer of the oropharynx that varies between 10% and 70% by geographic area and the burden of cancer caused by tobacco use. Most HPV infection is harmless and clears spontaneously but persistent infection with high risk HPV (notably type 16) is one of the most powerful human carcinogens. HPV disrupts normal cell cycle control promoting uncontrolled cell division and the accumulation of genetic damage. Two effective prophylactic vaccines composed of HPV-16/18 and HPV-16/18/6/11 virus like particles have been introduced in many developed countries as a primary prevention strategy. HPV testing is clinically valuable for secondary prevention in triaging low grade cytology and as a test of cure following treatment. More sensitive than cytology, primary screening by HPV testing could allow screening intervals to be extended. If these prevention strategies can be implemented in developing countries, many thousands of lives could be saved. HIV-positive individuals are at increased risk of HPV infection and cancer sites associated with HPV. cART and cervical screening may avoid an increase in cervical cancer in sub-Saharan cancer.

HCV AND lymphoma: Genetic and epigenetic factors Zignego AL, Gragnani L, Fognani E, Piluso A Center for Systemic Manifestation of Hepatitis Viruses-University of Florence, IT
Over 180 million people worldwide are chronically infected with the hepatitis C virus (HCV). HCV infection is a major cause for hepatocellular carcinoma (HCC), moreover the association with B-cell lymphoproliferative disorders (LPDs) like mixed cryoglobulinemia (MC) or B-cell non-Hodgkin lymphoma (B-NHL) is undisputed. The mechanisms by which HCV contributes to LPD development are still poorly understood. Available data suggest that the viral infection may induce LPDs through a multifactorial and multistep process that involves the sustained activation of B cells, the abnormal and prolonged B cell survival, and genetic and/or epigenetic factors.
Concerning genetic factors, different authors reported an association between specific HLA clusters or B-cell activating factor promoter genotype and a higher risk of developing MC and lymphoma. In addition, the results of a large, ongoing genome wide association study (GWAS) will probably allow the identification of specific genetic profile of HCV patients with LPDs.
Furthermore, microRNAs (miRNAs) can give a major contribution to the pathogenesis of several neoplastic, lymphoproliferative diseases and it is conceivable their involvement in the pathogenesis of HCV-related LPDs. We recently showed that specific miRNAs were differently modulated in PBMCs from HCV patients who developed MC and/or NHL. In addition, HCV patients who developed HCC, showed a differential miRNAs regulation.
In conclusion, available data suggest that the genetic/ epigenetic analysis of HCV-related cancerogenesis is of great usefulness in both the pathogenetic and clinical/ translational areas possibly allowing the definition of diagnostic/prognostic markers for early detection of lymphatic or hepatic cancer. The most relevant transforming mechanisms is the down-regulation of two oncosuppressor genes: p53 and pRb. For their frequency of detection in invasive lesions some genotypes have been defined "high risk" and their carcinogenicity has been referred to (a) their capacity to induce persistent infections, for the efficacy to escape the host immune system, (b) the binding affinity of their E6/E7 oncoprotein to the respective oncosuppressors, and (c) their efficacy to induce genomic instability. In vivo risk of cancer progression, in particular transition from pre-invasive to invasive cancer lesions, and the subsequent frequency of developing metastatic lesions as well as responsivity to anticancer treatment, is due to specific virus-host interactions and genomic damages. As paradigm the better prognosis and higher responsivity to treatment of HPV-positive oropharyngeal cancers in comparison to HPV-negative cancers. Mutations in genes of the apoptotic pathway, particularly the TP53 gene, have been analyzed in cervical cancers. A significant higher mutation frequency of TP53 gene in the CAC adenocarcinoma (32 of 241; 13.3%) compared to SCC squamous cell carcinoma (39 of 657; 5.9%; P=0.0003, и(2) test) has been identified. Three codons (175, 248 and 273) were the most commonly mutated in both, codon 249 mainly in SCC, codon 282 only in CAC. The G to A and C to T transitions were the prevalent type of mutations in both SCC and CAC (48.7% and 53.5% of all mutations, respectively). The frequency of C to A transversion was relatively high only in CAC (25%), while the mirror mutation G to T was comparatively frequent in SCC (14.6%). Although animal polyomaviruses, such as SV40, have been critical models in cancer research for over one-half century, polyomaviruses have not-until recently--been linked to human cancer. Using digital transcriptome subtraction, Merkel cell polyomavirus (MCV) was discovered in 2008 to infect most Merkel cell carcinomas, the most severe form of skin cancer. Normally, MCV is an common and asymptomatic infection of the human skin. In Merkel cell tumors, however, the virus integrates and undergoes mutations that eliminate viral replication but allow continued expression of viral oncogenes. MCV and MCC reveal a new model for carcinogenesis in which a rare combination of xenomutations to healthy skin flora initiate a deadly cancer. Using new sequencing technologies, the causes previouslyunsuspected viral cancers can be uncovered and clues for new rational drug therapies can be designed. Human herpesvirus 8 is associated with the development of primary effusion lymphoma (PEL), an aggressive non-Hodgkin's lymphoma characterized by the proliferation of the malignant lymphocytes almost exclusively in large serous cavities. The mechanisms involved in the preferential tropism for serous cavities and in the aggressive course of PEL remain to be fully clarified. To study the role of host microenvironment in PEL progression, we previously compared the antineoplastic activity of a murine interferon -expressing lentiviral vector (mIFN--LV) to that of a human IFN--LV in a murine model of peritoneal PEL. We demonstrated that in vivo targeting of the murine microenvironment showed an antineoplastic activity comparable to that observed with the hIFN--LV. These findings highlighted the relevant role of body cavity environment in PEL growth and indicated that modulation of microenvironment may impair PEL growth in vivo. By using cocultures of PEL cell lines with human mesothelial cells (HMC), we mimicked the interactions existing in body cavities to analyze the mechanisms involved in PEL progression. PEL cells induced a myofibroblastic morphology in HMC, paralleled by an expression profile indicative of the occurrence of epithelial-mesenchymal transition (EMT). Moreover, HMC increased proliferation and resistance to apoptosis of PEL cells. These data indicate that PEL cells induce EMT in HMC and fibrosis of serous membranes. In turn, HMC modulate PEL cell turnover, thus providing a milieu favorable to PEL progression. These findings open new perspectives into the mechanisms involved in PEL progression and may indicate new targets for PEL treatment. Human T-cell leukemia virus type 1 (HTLV-1) mainly transmits by cell-to-cell contact. Therefore, this virus induces proliferation of infected cells to facilitate transmission. In HTLV-1 infected individuals, infected cells proliferate in vivo. This proliferation of infected cells leads to the development of adult T-cell leukemia (ATL) and inflammatory diseases including HTLV-1 associated myelopathy (HAM). HTLV-1 bZIP factor (HBZ) is consistently expressed in ATL cells and HTLV-1 infected cells, and plays a critical role in their proliferation. Transgenic expression of HBZ causes T-cell lymphoma and inflammatory diseases, indicating that HBZ is critical for the pathogenesis of HTLV-1.

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We analyzed the function of HBZ and found that it possessed opposite functions to Tax in many pathways. We found that Tax could activate the classical Wnt pathway while HBZ inhibited it by interacting with TCF1/LEF1. The canonical Wnt pathway is activated in the thymus, and its transcription factors, TCF1/ LEF1, suppressed the viral gene transcription by Tax. Conversely, the canonical Wnt pathway is suppressed in the peripheral T cells in which the non-canonical Wnt pathway is activated. HBZ enhanced the expression of Wnt5a, which is a ligand in non-canonical Wnt signaling. Knockdown of Wnt5a suppressed the proliferation and migration of ATL cells. Thus, activation of the non-canonical Wnt pathway by HBZ is linked with proliferation of ATL cells.
These findings suggest that HTLV-1 is adapted to, not thymic T cells, but peripheral T cells. HBZ modulates the intracellular environment suitable for HTLV-1 replication and proliferation of infected cells.