Immunization with HIV Gag targeted to dendritic cells followed by recombinant New York vaccinia virus induces robust T-cell immunity in nonhuman primates.

Protein vaccines, if rendered immunogenic, would facilitate vaccine development against HIV and other pathogens. We compared in nonhuman primates (NHPs) immune responses to HIV Gag p24 within 3G9 antibody to DEC205 ("DEC-HIV Gag p24"), an uptake receptor on dendritic cells, to nontargeted protein, with or without poly ICLC, a synthetic double stranded RNA, as adjuvant. Priming s.c. with 60 μg of both HIV Gag p24 vaccines elicited potent CD4(+) T cells secreting IL-2, IFN-γ, and TNF-α, which also proliferated. The responses increased with each of three immunizations and recognized multiple Gag peptides. DEC-HIV Gag p24 showed better cross-priming for CD8(+) T cells, whereas the avidity of anti-Gag antibodies was ∼10-fold higher with nontargeted Gag 24 protein. For both protein vaccines, poly ICLC was essential for T- and B-cell immunity. To determine whether adaptive responses could be further enhanced, animals were boosted with New York vaccinia virus (NYVAC)-HIV Gag/Pol/Nef. Gag-specific CD4(+) and CD8(+) T-cell responses increased markedly after priming with both protein vaccines and poly ICLC. These data reveal qualitative differences in antibody and T-cell responses to DEC-HIV Gag p24 and Gag p24 protein and show that prime boost with protein and adjuvant followed by NYVAC elicits potent cellular immunity.

Impact of donor and recipient IL28B rs12979860 genotypes on hepatitis C virus liver graft reinfection.

BACKGROUND & AIMS: Recent studies have described a major impact of genetic variations near the IL28B gene on the natural course and outcome of antiviral therapy in chronic hepatitis C. We therefore, aimed to explore the impact of donor and recipient genotypes of these polymorphisms on hepatitis C virus (HCV) liver graft reinfection. METHODS: Donor and recipient genotypes of IL28B rs12979860C>T single nucleotide polymorphism were determined in 91 patients with HCV liver graft reinfection, 47 of whom were treated with pegylated interferon-α (PEG-IFN-α) and ribavirin. IL28B genetic polymorphisms were correlated with the natural course and treatment outcome of recurrent hepatitis C. RESULTS: Patients requiring liver transplantation due to end-stage chronic hepatitis C appeared to be selected toward the adverse genotypes rs12979860 CT/TT compared to non-transplanted HCV-infected patients (p=0.046). Patients with the donor genotype rs12979860 CC had higher peak ALT and HCV RNA serum concentrations than those with CT/TT (p=0.04 and 0.06, respectively). No association was observed between ALT/HCV RNA serum concentrations and recipient genotypes (p>0.3). More important, donor IL28B rs12979860 CC vs. CT/TT genotypes were associated with rapid, complete early, and sustained virologic response (RVR, cEVR, SVR) to treatment with PEG-IFN-α and ribavirin (p=0.003, 0.0012, 0.008, respectively), but weaker associations of recipient genotypes with RVR, cEVR, and SVR were observed as well (p=0.0046, 0.115, 0.118, respectively). CONCLUSIONS: We provide evidence for a dominant, but not exclusive impact of the donor rather than the recipient IL28B genetic background on the natural course and treatment outcome of HCV liver graft reinfection.

HLA class I - associated immunodominance affects CTL responsiveness to an ESO recombinant protein tumor antigen vaccine.

PURPOSE: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders. EXPERIMENTAL DESIGN: We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL. RESULTS: CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them. CONCLUSIONS: Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.

Comparison of Immunogenicity in Rhesus Macaques of Transmitted-Founder, HIV-1 Group M Consensus, and Trivalent Mosaic Envelope Vaccines Formulated as a DNA Prime, NYVAC, and Envelope Protein Boost.

An effective human immunodeficiency virus type 1 (HIV-1) vaccine must induce protective antibody responses, as well as CD4(+) and CD8(+) T cell responses, that can be effective despite extraordinary diversity of HIV-1. The consensus and mosaic immunogens are complete but artificial proteins, computationally designed to elicit immune responses with improved cross-reactive breadth, to attempt to overcome the challenge of global HIV diversity. In this study, we have compared the immunogenicity of a transmitted-founder (T/F) B clade Env (B.1059), a global group M consensus Env (Con-S), and a global trivalent mosaic Env protein in rhesus macaques. These antigens were delivered using a DNA prime-recombinant NYVAC (rNYVAC) vector and Env protein boost vaccination strategy. While Con-S Env was a single sequence, mosaic immunogens were a set of three Envs optimized to include the most common forms of potential T cell epitopes. Both Con-S and mosaic sequences retained common amino acids encompassed by both antibody and T cell epitopes and were central to globally circulating strains. Mosaics and Con-S Envs expressed as full-length proteins bound well to a number of neutralizing antibodies with discontinuous epitopes. Also, both consensus and mosaic immunogens induced significantly higher gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) responses than B.1059 immunogen. Immunization with these proteins, particularly Con-S, also induced significantly higher neutralizing antibodies to viruses than B.1059 Env, primarily to tier 1 viruses. Both Con-S and mosaics stimulated more potent CD8-T cell responses against heterologous Envs than did B.1059. Both antibody and cellular data from this study strengthen the concept of using in silico-designed centralized immunogens for global HIV-1 vaccine development strategies. IMPORTANCE: There is an increasing appreciation for the importance of vaccine-induced anti-Env antibody responses for preventing HIV-1 acquisition. This nonhuman primate study demonstrates that in silico-designed global HIV-1 immunogens, designed for a human clinical trial, are capable of eliciting not only T lymphocyte responses but also potent anti-Env antibody responses.

A Lead-In with Silibinin Prior to Triple-Therapy Translates into Favorable Treatment Outcomes in Difficult-To-Treat HIV/Hepatitis C Coinfected Patients.

BACKGROUND: The efficacy of first-generation protease inhibitor based triple-therapy against hepatitis C virus (HCV) infection is limited in HIV/HCV-coinfected patients with advanced liver fibrosis and non-response to previous peginterferon-ribavirin. These patients have a low chance of achieving a sustained virologic response (SVR) using first generation triple-therapy, with a success rate of only 20%. We investigated the efficacy and safety of lead-in therapy with intravenous silibinin followed by triple-therapy in this difficult-to-treat patient group. METHODOLOGY: Inclusion criteria were HIV/HCV coinfection with advanced liver fibrosis and documented previous treatment failure on peginterferon-ribavirin. The intervention was a lead-in therapy with intravenous silibinin 20 mg/kg/day for 14 days, followed by triple-therapy (peginterferon-ribavirin and telaprevir) for 12 weeks, and peginterferon-ribavirin alone for 36 weeks. Outcome measurements were HCV-RNA after silibinin lead-in and during triple-therapy, SVR data at week 12, and safety and tolerability of silibinin. RESULTS: We examined sixteen HIV/HCV-coinfected patients with previous peginterferon-ribavirin failure, of whom 14 had a fibrosis grade METAVIR ≥F3. All were on successful antiretroviral therapy. Median (IQR) HCV-RNA decline after silibinin therapy was 2.65 (2.1-2.8) log10 copies/mL. Fifteen of sixteen patients (94%) had undetectable HCV RNA at weeks 4 and 12, eleven patients (69%) showed end-of-treatment response (i.e., undetectable HCV-RNA at week 48), and ten patients (63%) reached SVR at week 12 (SVR 12). Six of the sixteen patients (37%) did not reach SVR 12: One patient had rapid virologic response (RVR) (i.e., undetectable HCV-RNA at week 4) but stopped treatment at week 8 due to major depression. Five patients had RVR, but experienced viral breakthroughs at week 21, 22, 25, or 32, or a relapse at week 52. The HIV RNA remained below the limit of detection in all patients during the complete treatment period. No serious adverse events and no significant drug-drug interactions were associated with silibinin. CONCLUSION: A lead-in with silibinin before triple-therapy was safe and highly effective in difficult-to-treat HIV/HCV coinfected patients, with a pronounced HCV-RNA decline during the lead-in phase, which translates into 63% SVR. An add-on of intravenous silibinin to standard of care HCV treatment is worth further exploration in selected difficult-to-treat patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT01816490.

Safety and Immunogenicity of a Recombinant Adenovirus Serotype 35-Vectored HIV-1 Vaccine in Adenovirus Serotype 5 Seronegative and Seropositive Individuals.

BACKGROUND: Recombinant adenovirus serotype 5 (rAd5)-vectored HIV-1 vaccines have not prevented HIV-1 infection or disease and pre-existing Ad5 neutralizing antibodies may limit the clinical utility of Ad5 vectors globally. Using a rare Ad serotype vector, such as Ad35, may circumvent these issues, but there are few data on the safety and immunogenicity of rAd35 directly compared to rAd5 following human vaccination. METHODS: HVTN 077 randomized 192 healthy, HIV-uninfected participants into one of four HIV-1 vaccine/placebo groups: rAd35/rAd5, DNA/rAd5, and DNA/rAd35 in Ad5-seronegative persons; and DNA/rAd35 in Ad5-seropositive persons. All vaccines encoded the HIV-1 EnvA antigen. Antibody and T-cell responses were measured 4 weeks post boost immunization. RESULTS: All vaccines were generally well tolerated and similarly immunogenic. As compared to rAd5, rAd35 was equally potent in boosting HIV-1-specific humoral and cellular immunity and responses were not significantly attenuated in those with baseline Ad5 seropositivity. Like DNA, rAd35 efficiently primed rAd5 boosting. All vaccine regimens tested elicited cross-clade antibody responses, including Env V1/V2-specific IgG responses. CONCLUSIONS: Vaccine antigen delivery by rAd35 is well-tolerated and immunogenic as a prime to rAd5 immunization and as a boost to prior DNA immunization with the homologous insert. Further development of rAd35-vectored prime-boost vaccine regimens is warranted.

A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is attenuated and immunogenic for calves

Bovine herpesvirus 5 (BoHV-5) is an important pathogen of cattle in South America and efforts have been made to produce safer and more effective vaccines. In addition to afford protection, herpesvirus vaccines should allow serological differentiation of vaccinated from naturally, latently infected animals. We previously reported the construction and characterization in vitro of a double mutant BoHV-5 (BoHV-5gE/TK Δ) lacking the genes encoding thymidine kinase (tk) for attenuation, and glycoprotein E (gE) as the antigenic marker, as a vaccine candidate strain (Brum et al. 2010a). The present article reports an investigation on the attenuation and immunogenicity of this recombinant in calves. In a first experiment, 80 to 90-day-old seronegative calves (n=6) inoculated intranasally with the recombinant (titer of 10(7.5)TCID50) shed virus in low to moderate titers in nasal secretions for up to 6 days, yet did not develop any respiratory, systemic or neurological signs of infection. At day 30 post-infection (pi) all calves had BoHV-5 specific neutralizing (VN) antibodies in titers of 4 to 8 and were negative for anti-gE antibodies in a commercial ELISA test. Administration of dexamethasone (0.1mg/kg/day during 5 days) to four of these calves at day 42 pi did not result in virus shedding or increase in VN titers, indicating lack of viral reactivation. Secondly, a group of 8-month-old calves (n=9) vaccinated intramuscularly (IM) with the recombinant virus (10(7.5)TCID50/animal) did not shed virus in nasal secretions, remained healthy and developed VN titers from 2 to 8 at day 42 post-vaccination (pv), remaining negative for gE antibodies. Lastly, 21 calves (around 10 months old) maintained under field conditions were vaccinated IM with the recombinant virus (titer of 10(7.3)TCID50). All vaccinated animals developed VN titers from 2 to 16 at day 30 pv. A boost vaccination performed at day 240 pv resulted in a rapid and strong anamnestic antibody response, with VN titers reaching from 16 to 256 at day 14 post-booster. Again, serum samples remained negative for gE antibodies. Selected serum samples from vaccinated animals showed a broad VN activity against nine BoHV-5 and eight BoHV-1 field isolates. These results show that the recombinant virus is attenuated, immunogenic for calves and induces an antibody response differentiable from that induced by natural infection. Thus, the recombinant BoHV-5gE/TKΔ is an adequate candidate strain for a modified live vaccine.

New vaccine strategies against enterotoxigenic Escherichia coli: II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine

The induction of systemic (IgG) and mucosal (IgA) antibody responses against the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC) was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

Prevalence of hepatitis C virus (HCV) infection and HCV genotypes of hemodialysis patients in Salvador, Northeastern Brazil

Hepatitis C virus (HCV) infection has been identified as the major cause of chronic liver disease among patients on chronic hemodialysis (HD), despite the important reduction in risks obtained by testing candidate blood donors for anti-HCV antibodies and the use of recombinant erythropoietin to treat anemia. A cross-sectional study was performed to estimate the prevalence of HCV infection and genotypes among HD patients in Salvador, Northeastern Brazil. Anti-HCV seroprevalence was determined by ELISA in 1243 HD patients from all ten different dialysis centers of the city. HCV infection was confirmed by RT-PCR and genotyping was performed by restriction fragment length polymorphism. Anti-HCV seroprevalence among HD patients was 10.5% (95% CI: 8.8-12.3) (Murex anti-HCV, Abbott Murex, Chicago, IL, USA). Blood samples for qualitative HCV detection and genotyping were collected from 125/130 seropositive HD patients (96.2%). HCV-RNA was detected in 92/125 (73.6%) of the anti-HCV-positive patients. HCV genotype 1 (77.9%) was the most prevalent, followed by genotype 3 (10.5%) and genotype 2 (4.6%). Mixed infections of genotypes 1 and 3 were found in 7.0% of the total number of patients. The present results indicate a significant decrease in anti-HCV prevalence from 23.8% detected in a study carried out in 1994 to 10.5% in the present study. The HCV genotype distribution was closely similar to that observed in other hemodialysis populations in Brazil, in local candidate blood donors and in other groups at risk of transfusion-transmitted infection.

Recombinant vaccines and the development of new vaccine strategies

Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.

Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

Induction of neutralizing antibodies in mice immunized with an amino-terminal polypeptide of Streptococcus mutans P1 protein produced by a recombinant Bacillus subtilis strain

The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P1(39-512)) derived from the S. mutans strain UA159. Purified P1(39-512) reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P1(39-512) induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P1(39-512) antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P1(39-512) eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P1(39-512), expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.