Homologous Neutralizing Antibodies Research Articles

Phase 1 dose-escalation trial evaluating a group 2 influenza hemagglutinin stabilized stem nanoparticle vaccine

The relative conservation of the influenza hemagglutinin (HA) stem compared to that of the immunodominant HA head makes the HA stem an attractive target for broadly protective influenza vaccines. Here we report the first-in-human, dose-escalation, open-label trial (NCT04579250) evaluating an unadjuvanted group 2 stabilized stem ferritin nanoparticle vaccine based on the H10 A/Jiangxi-Donghu/346/2013 influenza HA, H10ssF, in healthy adults. Participants received a single 20 mcg dose (n = 3) or two 60 mcg doses 16 weeks apart (n = 22). Vaccination with H10ssF was safe and well tolerated with only mild systemic and local reactogenicity reported. No serious adverse events occurred. Vaccination significantly increased homologous H10 HA stem binding and neutralizing antibodies at 2 weeks after both first and second vaccinations, and these responses remained above baseline at 40 weeks. Heterologous H3 and H7 binding antibodies also significantly increased after each vaccination and remained elevated throughout the study. These data indicate that the group 2 HA stem nanoparticle vaccine is safe and induces stem-directed binding and neutralizing antibodies.

Wa-VP4* as a candidate rotavirus vaccine induced homologous and heterologous virus neutralizing antibody responses in mice, pigs, and cynomolgus monkeys

Group A rotavirus (RVA) is the primary etiological agent of acute gastroenteritis (AGE) in children under 5 years of age. Despite the global implementation of vaccines, rotavirus infections continue to cause over 120,000 deaths annually, with a majority occurring in developing nations. Among infants, the P[8] rotavirus strain is the most prevalent and can be categorized into four distinct lineages. In this investigation, we expressed five VP4(aa26-476) proteins from different P[8] lineages of human rotavirus in E. coli and assessed their immunogenicity in rabbits. Among the different P[8] strains, the Wa-VP4 protein, derived from the MT025868.1 strain of the P[8]-1 lineage, exhibited successful purification in a highly homogeneous form and significantly elicited higher levels of neutralizing antibodies (nAbs) against both homologous and heterologous rotaviruses compared to other VP4 proteins derived from different P[8] lineages in rabbits. Furthermore, we assessed the immunogenicity of the Wa-VP4 protein in mice, pigs, and cynomolgus monkeys, observing that it induced robust production of nAbs in all animals. Interestingly, there was no significant difference between in nAb titers against homologous and heterologous rotaviruses in pigs and mankeys. Collectively, these findings suggest that the Wa-VP4* protein may serve as a potential candidate for a rotavirus vaccine.

Efficacy of a porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) natural recombinant against a heterologous PRRSV-1 isolate both clustered within the subgroup of BJEU06-1-like isolates

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) has been prevalent in more than 20 provinces of China. However, no PRRSV-1-specific vaccine is commercially available in China. To evaluate the feasibility of using a low virulent PRRSV-1 isolate against potential outbreaks caused by virulent Chinese PRRSV-1 isolates, here we evaluated the efficacy of a low virulent PRRSV-1 HLJB1 strain isolated in 2014 as live vaccine against a virulent PRRSV-1 SD1291 strain isolated in 2022. Genome-based phylogenetic analysis showed that both HLJB1 and SD1291 were grouped within BJEU06–1-like isolates. However, they shared only 85.27% genomic similarity. Piglet inoculation and challenge study showed that HLJB1 inoculation could reduce viremia but did not significantly alleviate clinical signs and tissue lesions. Virus neutralization test indicated that HLJB1 inoculation could induce homologous neutralizing antibodies (NAbs) but no heterologous NAbs at 42 dpi. In addition, flow cytometric analyses showed that no memory T follicular helper (Tfh) cells against SD1291 and SD1291-specific IFN-γ secreting cells were induced by HLJB1 pre-inoculation. These results supported that HLJB1 inoculation only provides partial cross-protection against SD1291 infection even though they are clustered within the same PRRSV-1 subgroup, which is closely related to the failure in conferring cross-protective adaptive immune responses.

Fc-mediated pan-sarbecovirus protection after alphavirus vector vaccination

Group 2B β-coronaviruses (sarbecoviruses) have caused regional and global epidemics in modern history. Here, we evaluate the mechanisms of cross-sarbecovirus protective immunity, currently less clear yet important for pan-sarbecovirus vaccine development, using a panel of alphavirus-vectored vaccines covering bat to human strains. While vaccination does not prevent virus replication, it protects against lethal heterologous disease outcomes in both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clade 2 bat sarbecovirus challenge models. The spike vaccines tested primarily elicit a highly S1-specific homologous neutralizing antibody response with no detectable cross-virus neutralization. Rather, non-neutralizing antibody functions, mechanistically linked to FcgR4 and spike S2, mediate cross-protection in wild-type mice. Protection is lost in FcR knockout mice, further supporting a model for non-neutralizing, protective antibodies. These data highlight the importance of FcR-mediated cross-protective immune responses in universal pan-sarbecovirus vaccine designs.

Systemic Homologous Neutralizing Antibodies Are Inadequate for the Evaluation of Vaccine Protective Efficacy against Coinfection by High Virulent PEDV and PRRSV.

ABSTRACTG2 porcine epidemic diarrhea virus (G2 PEDV) and highly pathogenic porcine reproductive and respiratory syndrome virus 2 (HP-PRRSV2) are two of the most prevalent swine pathogens in China’s swine herds, and their coinfection occurs commonly. Several PED and PRRS vaccines have been utilized in China for decades, and systemic homologous neutralizing antibodies (shnAbs) in serum are frequently used to evaluate the protective efficacy of PED and PRRS vaccines. To develop a vaccine candidate against G2 PEDV and HP-PRRSV2 coinfection, in this study, we generated a chimeric virus (rJSTZ1712-12-S) expressing S protein of G2 PEDV using an avirulent HP-PRRSV2 rJSTZ1712-12 infectious clone as the viral vector. The rJSTZ1712-12-S strain has similar replication efficacies as the parental rJSTZ1712-12 virus. In addition, animal inoculation indicated that rJSTZ1712-12-S is not pathogenic to piglets and can induce shnAbs against both G2 PEDV and HP-PRRSV2 isolates after prime-boost immunization. However, passive transfer study in neonatal piglets deprived of sow colostrum showed that rJSTZ1712-12-S-induced shnAbs may only decrease PEDV and PRRSV viremia but cannot confer sufficient protection against dual challenge of high virulent G2 PEDV XJ1904-34 strain and HP-PRRSV2 XJ17-5 isolate. Overall, this study provides the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for the evaluation of protective efficacy of PED and PRRS bivalent vaccine (especially for the PED vaccine).IMPORTANCE Porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) coinfection occurs commonly and can synergistically reduce feed intake and pig growth. Vaccination is an effective strategy utilized for PED and PRRS control, and systemic homologous neutralizing antibodies (shnAbs) in serum are commonly used for protective efficacy evaluation of PED and PRRS vaccines. Currently, no commercial vaccine is available against PEDV and PRRSV coinfection. This study generated a chimeric vaccine candidate against the coinfection of prevalent PEDV and PRRSV in China. The chimeric strain can induce satisfied shnAbs against both PEDV and PRRSV after prime-boost inoculation in pigs. But the shnAbs cannot confer sufficient protection against PEDV and PRRSV coinfection in neonatal piglets. To the best of our knowledge, these findings provide the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for evaluating PED and PRRS bivalent vaccine protective efficacy.

Long-lasting heterologous antibody responses after sequential vaccination with A/Indonesia/5/2005 and A/Vietnam/1203/2004 pre-pandemic influenza A(H5N1) virus vaccines

BackgroundAvian influenza A(H5N1) viruses have caused sporadic infections in humans and thus they pose a significant global health threat. Among symptomatic patients the case fatality rate has been ca. 50%. H5N1 viruses exist in multiple clades and subclades and several candidate vaccines have been developed to prevent A(H5N1) infection as a principal measure for preventing the disease. MethodsSerum antibodies against various influenza A(H5N1) clade viruses were measured in adults by ELISA-based microneutralization and haemagglutination inhibition tests before and after vaccination with two different A(H5N1) vaccines in 2009 and 2011. ResultsTwo doses of AS03-adjuvanted A/Indonesia/5/2005 vaccine induced good homologous but poor heterologous neutralizing antibody responses against different clade viruses. However, non-adjuvanted A/Vietnam/1203/2004 booster vaccination in 2011 induced very strong and long-lasting homologous and heterologous antibody responses while homologous response remained weak in naïve subjects. ConclusionsSequential vaccination with two different A(H5N1) pre-pandemic vaccines induced long-lasting high level cross-clade immunity against influenza A(H5N1) strains, thus supporting a prime-boost vaccination strategy in pandemic preparedness plans.

Comparative humoral immune response against cytopathic or non-cytopathic bovine viral diarrhea virus infection

Bovine viral diarrhea virus (BVDV) infection causes immune dysfunction. The current study investigated the effect of cytopathic (cp) or noncytopathic (ncp) strains of BVDV on immunomodulation by the levels of total serum immunoglobulin G (IgG), the IgG1, IgG2, BVDV neutralizing antibodies and total white blood cell (WBC) count. Twenty (20) BVDV seronegative dairy calves (5–6 months old) were divided in two groups of ten. The animals were infected with either a cp or ncp virus isolated from the same animal (ncp BVDV1b-TGAN or cp BVDV1b-TGAC). One group of 10 was infected with ncp TGAN while the other group of 10 was infected with cp TGAC. Calves infected with cp BVDV had a significant decrease in total IgG as well as IgG1 concentration at 7 days post infection (DPI) that recovered by 21 DPI (total IgG) and 35 DPI (IgG1), respectively. There was no effect of ncp BVDV infection on total IgG concentration in the first 7 days of infection (DOI); however, IgG1 concentration was significantly reduced and IgG2 concentration was significantly increased at 7 DOI. At 35 DPI, ncp TGAN-infected calves had significantly higher total IgG, IgG1 as well as IgG2 compared to cp TGAC-infected calves. Ncp BVDV induced higher BVDV homologous and heterologous neutralizing antibodies compared to the cp BVDV strain. Calves infected with ncp BVDV had significantly reduced WBC counts at 7 DPI that recovered by 14 DPI. Overall, these findings indicate that humoral immunosuppression occurs early following BVDV infection with the largest effect on IgG1 levels.

Safety and long-lasting immunity of the combined administration of a modified-live virus vaccine against porcine reproductive and respiratory syndrome virus 1 and an inactivated vaccine against porcine parvovirus and Erysipelothrix rhusiopathiae in breeding pigs

BackgroundIn the field, vaccination schedules based on modified-live virus (MLV) vaccines administered twice in gilts and every three to four months in sows are commonly used to immunize breeding herds against porcine reproductive and respiratory virus (PRRSV). Breeding sows are repeatedly vaccinated against several other agents. Thus, the combined administration of vaccines for their simultaneous use can simplify such complex immunization schedules. Here, we evaluated the safety and long-term immunity of the authorized combined administration of a PRRSV MLV vaccine and an inactivated vaccine against porcine parvovirus (PPV) and Erysipelothrix rhusiopathiae for their simultaneous use.Six-month-old naïve healthy gilts were vaccinated at day 0 and revaccinated at days 21 and 147, mimicking the abovementioned vaccination schedule. Systemic and local reactions, as well as body temperature, were measured. The excretion of PRRSV1 MLV was evaluated in oral fluids. Humoral responses against the three antigens were measured by ELISA. For PRRSV, homologous neutralizing antibodies (NAs) and homologous and heterologous cell-mediated immunity (CMI) were also assessed.ResultsThe combined administration of the tested vaccines, applied according to the manufacturer’s instructions, was safe based on all evaluated parameters. Overall, we detected antibodies against PPV and PRRSV in all vaccinated pigs already after the first vaccination, whereas antibodies against E. rhusiopathiae were observed in all animals after revaccination. After subsequent revaccinations, we observed boosts for the humoral response for PPV at days 28 and 154 and at day 154 for E. rhusiopathiae. No boosts were detected during the experiment by PRRSV ELISA. In all vaccinated animals, homologous NAs against MLV were already detected before revaccination (day 21). After revaccination, there was a boost with mean titres of homologous NAs remaining constant thereafter. Concerning CMI, PRRSV-specific IFN-γ-secreting cells were already detected at day 21 for all evaluated strains and we observed boosts for all PRRSV1 strains after revaccination and recall revaccination.ConclusionsWe showed that the combined administration of tested vaccines described here using a vaccination schedule against PRRSV commonly implemented for breeding pigs in the field is safe and induces long-lasting humoral and cellular immunity against PRRSV, PPV, and E. rhusiopathiae.

Versatility of the adenovirus-vectored foot-and-mouth disease vaccine platform across multiple foot-and-mouth disease virus serotypes and topotypes using a vaccine dose representative of the AdtA24 conditionally licensed vaccine

We investigated the serotype- and topotype versatility of a replication-deficient human adenovirus serotype 5 vectored foot-and-mouth disease (FMD) vaccine platform (AdtFMD). Sixteen AdtFMD recombinant subunit monovalent vaccines targeting twelve distinct FMD virus (FMDV) serotype/topotypes in FMD Regional Pools I-VII were constructed. The AdtA24 serotype conditionally licensed vaccine served as the basis for vaccine design and target dose for cattle clinical trials. Several vaccines contained an additional RGD motif genetic insertion in the adenovector fiber knob, and/or a full-length 2B gene insertion in the FMDV P1 gene cassette. In 13 of the 22 efficacy studies conducted, naïve control and AdtFMD vaccinated cattle were challenged intradermolingually at 2 weeks post-vaccination using a FMDV strain homologous to the AdtFMD vaccine strain. Each of the 16 AdtFMD vaccines were immunogenic based on the presence of homologous neutralizing antibodies in the serum of approximately 90% of total vaccinates (n = 375) on the day of challenge. Importantly, for 75% of vaccines tested, the effective dose that conferred 100% protection against clinical FMD was identical to or in some cases lower than, the minimum protective dose for the conditionally licensed AdtA24 vaccine formulated with ENABL® adjuvant. Results also confirmed the capability of the AdtFMD vaccine platform to differentiate infected from vaccinated animals (DIVA) across the five FMDV serotypes evaluated. Collectively, this comprehensive set of FMD cattle vaccine dose ranging studies highlights the serotype- and topotype versatility of the AdtFMD vaccine platform for further development, licensure, and application in FMD outbreak control and disease eradication efforts.

Neutralizing Antibody Responses to Homologous and Heterologous H1 and H3 Influenza A Strains After Vaccination With Inactivated Trivalent Influenza Vaccine Vary With Age and Prior-year Vaccination.

Prior influenza immunity influences the homologous neutralizing antibody responses elicited by inactivated influenza vaccines (IIV), but neutralizing antibody responses to heterologous strains have not been extensively characterized. We analyzed neutralizing antibody titers in individuals aged 1-88 who received the 2009-2010 season IIV before infection by or vaccination against the 2009 pandemic H1N1 virus. Neutralization titers to homologous and heterologous past, recent, and advanced H1 and H3 strains, as well as H2, H5, and H7 strains, were measured using influenza hemagglutinin pseudoviruses. We performed exploratory analyses based on age, prior-year IIV, and prevaccination titer, without controlling for Type I errors. IIV elicited neutralizing antibodies to past and advanced H1 and H3 strains, as well as to an H2 strain in individuals who were likely infected early in life. The neutralization of avian subtype viruses was rare, and there was no imprinting of neutralization responses to novel avian subtype viruses based on the influenza group. Compared to adults, children had higher seroresponse rates to homologous and heterologous strains, and their sera generated larger antigenic distances among strains. Seroresponse rates to homologous and heterologous strains were lower in subjects vaccinated with prior-year IIV, though postimmunization titers were generally high. IIV elicited neutralizing antibodies to heterologous H1 and H3 strains in all ages groups, but titers and seroresponse rates were usually higher in children. Prior-year vaccination with the same strains tended to blunt IIV neutralization responses to all strains in young and old age groups, yet postimmunization titers were high.

Cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection

Because porcine reproductive and respiratory syndrome virus (PRRSV) exhibits extensive genetic variation among field isolates, characterizing the extent of cross reactivity of immune responses, and most importantly cell-mediated immunity (CMI), could help in the development of broadly cross-protective vaccines. We infected 12 PRRSV-naïve pigs with PRRSV strain FL12 and determined the number of interferon (IFN)-γ secreting cells (SC) by ELISpot assay using ten type 2 and one type 1 PRRSV isolates as recall antigens. The number of IFN-γ SC was extremely variable among animals, and with exceptions, late to appear. Cross reactivity of IFN-γ SC among type 2 isolates was broad, and we found no evidence of an association between increased genetic distance among isolates and the intensity of the CMI response. Comparable to IFN-γ SC, total antibodies evaluated by indirect immunofluorescence assay (IFA) were cross reactive, however, neutralizing antibody titers could only be detected against the strain used for infection. Finally, we observed a moderate association between homologous IFN-γ SC and neutralizing antibodies.

High-Density Array of Well-Ordered HIV-1 Spikes on Synthetic Liposomal Nanoparticles Efficiently Activate B Cells.

A major step toward an HIV-1 vaccine is an immunogen capable of inducing neutralizing antibodies. Envelope glycoprotein (Env) mimetics, such as the NFL and SOSIP designs, generate native-like, well-ordered trimers and elicit tier 2 homologous neutralization (SOSIPs). We reasoned that the display of well-ordered trimers by high-density, particulate array would increase B cell activation compared to soluble trimers. Here, we present the design of liposomal nanoparticles displaying well-ordered Env spike trimers on their surface. Biophysical analysis, cryo- and negative stain electron microscopy, as well as binding analysis with a panel of broadly neutralizing antibodies confirm a high-density, well-ordered trimer particulate array. The Env-trimer-conjugated liposomes were superior to soluble trimers in activating B cells exvivo and germinal center B cells invivo. In addition, the trimer-conjugated liposomes elicited modest tier 2 homologous neutralizing antibodies. The trimer-conjugated liposomes represent a promising initial lead toward the development of more effective HIV vaccine immunogens.

Safety and immunogenicity of a parenterally administered rotavirus VP8 subunit vaccine in healthy adults

BackgroundThe P2-VP8 subunit vaccine for the prevention of rotavirus gastroenteritis is comprised of a truncated VP8 subunit protein from the rotavirus Wa strain (G1[P8]) fused to the tetanus toxin P2 epitope, and adsorbed on aluminum hydroxide for intramuscular administration. MethodsThree groups of 16 adults were randomized to receive three injections of P2-VP8 (12) or placebo (4) at doses of 10, 30 or 60μg of vaccine. IgG and IgA antibodies to P2-VP8 were assessed by ELISA in serum and lymphocyte supernatant (ALS). Serum samples were tested for neutralizing antibodies to homologous and heterologous strains of rotavirus. ResultsThe vaccine was well-tolerated. All vaccine recipients demonstrated significant IgA responses and all but one demonstrated IgG responses; in the 60μg cohort, geometric mean titers (GMTs) rose 70- and 80-fold for IgA and IgG, respectively. Homologous neutralizing antibody responses were observed in about half of participants in all three dose cohorts; in the 60μg cohort, GMTs against Wa rose from 128 to 992. Neutralizing antibody responses were robust to P[8] strains, moderate to P[4] strains and negligible to P[6] strains. ALS IgA responses were dose dependent. ConclusionsThe P2-VP8 subunit vaccine was well tolerated and evoked promising immune responses. Clinical trials registrationNCT01764256

Immunogenicity of recombinant VP2 proteins of all nine serotypes of African horse sickness virus

African horse sickness (AHS) is an equine disease with a mortality of up to 90% for susceptible horses. The causative agent AHS virus (AHSV) is transmitted by species of Culicoides. AHSV serogroup within the genus Orbivirus of the Reoviridae family consists of nine serotypes that show no or very limited cross-neutralization. Of the seven structural proteins (VP1-VP7) of AHSV, VP2 is the serotype specific protein, and the major target for neutralizing antibodies. In this report, recombinant VP2 proteins of all nine serotypes were expressed individually by the baculovirus expression system and the immunogenicity of each was studied by immunization of guinea pigs with single VP2 as well as with cocktails of VP2 proteins. Homologous neutralizing antibodies measured by 50% plaque reduction assay showed varying degrees (from 37 to 1365) of titers for different VP2 proteins. A low cross-neutralizing antibody titer was found for genetically related AHSV serotypes. Immunization with VP2 cocktails containing equal amounts of each of the VP2 proteins also triggered neutralizing antibodies albeit to lower titers (4-117) to each of the serotypes in the cocktail. This study is a first step to develop a VP2 subunit vaccine for AHS and our results indicate that VP2 subunit vaccines are feasible individually or in a multi-serotype cocktail.

Virus-like particle secretion and genotype-dependent immunogenicity of dengue virus serotype 2 DNA vaccine.

Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine candidates were evaluated for their immunogenicity, homologous and heterologous neutralizing (Nt) antibody titers, and cross-genotype protection in a murine model. The immunity elicited by our prototype vaccine candidate (Asian 1 genotype strain 16681) in mice was protective against viruses of other genotypes but not against virus of the Sylvatic genotype, whose emergence and potential risk after introduction into the human population have previously been demonstrated. The underlying mechanism of a lack of protection elicited by the prototype vaccine may at least be contributed by the absence of a flavivirus subgroup-cross-reactive, highly neutralizing monoclonal antibody 1B7-5-like epitope in DENV-2 of the Sylvatic genotype. The DENV DNA vaccine directs the synthesis and assembly of virus-like particles (VLPs) and induces immune responses similar to those elicited by live-attenuated vaccines, and its flexibility permits the fast deployment of vaccine to combat emerging viruses, such as Sylvatic genotype viruses. The enhanced VLP secretion obtained by replacement of ectodomain I-II (EDI-II) of the Cosmopolitan genotype vaccine construct (VD2-Cosmopolitan) with the Asian 1 EDI-II elicited significantly higher total IgG and Nt antibody titers and suggests a novel approach to enhance the immunogenicity of the DNA vaccine. A DENV vaccine capable of eliciting protective immunity against viruses of existing and emerging genotypes should be the focus of future DENV vaccine development.

Inclusion of a universal tetanus toxoid CD4+ T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines

Currently available live oral rotavirus vaccines, Rotarix® and RotaTeq®, are highly efficacious in developed countries. However, the immunogenicity and efficacy of such vaccines in some developing countries are low. We reported previously that bacterially-expressed rotavirus ΔVP8* subunit vaccine candidates with P[8], P[4] or P[6] specificity elicited high-titer virus neutralizing antibodies in animals immunized intramuscularly. Of note was the finding that antibodies induced with the P[8]ΔVP8* vaccine neutralized both homotypic P[8] and heterotypic P[4] rotavirus strains to high titer. To further improve its vaccine potential, a tetanus toxoid universal CD4+ T cell epitope P2 was introduced into P[8] or P[6]ΔVP8* construct. The resulting recombinant fusion proteins expressed in Escherichia coli were of high solubility and were produced with high yield. Two doses (10 or 20μg/dose) of the P2-P[8]ΔVP8* vaccine or P2-P[6]ΔVP8* vaccine with aluminum phosphate adjuvant elicited significantly higher geometric mean homologous neutralizing antibody titers than the vaccines without P2 in intramuscularly immunized guinea pigs. Interestingly, high levels of neutralizing antibody responses induced in guinea pigs with 3 doses of the P2-P[8]ΔVP8* vaccine persisted for at least 6 months. Furthermore, in the gnotobiotic piglet challenge study, three intramuscular doses (50μg/dose) of the P2-P[8]ΔVP8* vaccine with aluminum phosphate adjuvant significantly delayed the onset of diarrhea and significantly reduced the duration of diarrhea and the cumulative diarrhea score after oral challenge with virulent human rotavirus Wa (G1P[8]) strain. The P2-P[8]ΔVP8* vaccine induced serum virus neutralizing antibody and VP4-specific IgG antibody production prechallenge, and primed the pigs for higher antibody and intestinal and systemic virus-specific IFN-γ producing CD4+ T cell responses postchallenge. These two subunit vaccines could be used at a minimum singly or preferably in bivalent formulation to provide antigenic coverage of most of the G types of global importance.

Immunogenicity of low-pH treated whole viral influenza vaccine

Low pH treatment of influenza virus hemagglutinin (HA) exposes its relatively conserved stalk domain, suggesting a potential immunogen with capability to induce broader immune responses. Here, we describe characterization, immunogenicity, antigenicity, and protective immunity induced by low pH treated inactivated whole viral vaccine in comparison with the untreated vaccine. The acidic pH treated viral vaccine showed high susceptibility to proteolytic cleavage and low hemagglutination activity indicating conformational changes. Immunization of mice with low pH treated viral vaccine induced lower levels of homologous or heterologous virus-specific binding and neutralizing antibodies compared to the untreated vaccine. Also, low pH treated influenza viral antigen showed lower antigenicity compared to the untreated influenza viral antigen. Lower efficacy of cross-protection against heterosubtypic virus was observed in the low-pH treated vaccine group. The results provide evidence that there is a correlation between protective efficacy and the stability of vaccines.

Highly Efficient JFH1‐Based Cell‐Culture System for Hepatitis C Virus Genotype 5a: Failure of Homologous Neutralizing‐Antibody Treatment to Control Infection

Recently, a hepatitis C virus (HCV) cell-culture system was developed that employed strain JFH1 (genotype 2a), and JFH1-based intra- and intergenotypic recombinants now permit functional studies of the structural genes (Core, E1, and E2), p7, and NS2 of genotypes 1-4. The goal was to adapt the system to employ genotype 5. Huh7.5 cells infected with SA13/JFH1, containing Core-NS2 of strain SA13 (genotype 5a), were monitored for Core expression and for supernatant infectivity and HCV-RNA titers. Adaptive mutations of SA13/JFH1 were identified by sequence analysis of recovered genomes and reverse-genetic studies. Receptor blockage was performed with anti-CD81 and anti-SR-BI. For neutralization experiments, SA13/JFH1 or JFH1-based viruses of other genotypes were incubated with patient sera. SA13/JFH1 with NS2 and NS3 mutations yielded infectivity titers >10(5) TCID50/mL. Infection with SA13/JFH1 was inhibited by CD81 blocking and SR-BI blocking, respectively, and by preincubation with genotype 5a chronic-phase patient sera. Such sera had varying cross-genotype neutralization potential. However, preincubation and treatment with homologous neutralizing antibodies could not control SA13/JFH1 infection in culture. The SA13/JFH1 culture permits genotype 5a-specific studies of Core-NS2 function and interfering agents. The ability of HCV to spread in vivo during treatment with neutralizing antibodies was confirmed in vitro.

Characterization of Neutralizing Antibody Responses Elicited by Clade A Envelope Immunogens Derived from Early Transmitted Viruses

The vast majority of studies with candidate immunogens based on the human immunodeficiency virus envelope (Env) have been conducted with Env proteins derived from clade B viruses isolated during chronic infection. Whether non-clade B Env protein immunogens will elicit antibodies with epitope specificities that are similar to those of antibodies elicited by clade B Envs and whether the antibodies elicited by Envs derived from early transmitted viruses will be similar to those elicited by Envs derived from viruses isolated during chronic infection are currently unknown. Here we performed immunizations with four clade A Envs, cloned directly from the peripheral blood of infected individuals during acute infection, which differed in lengths and extents of glycosylation. The antibody responses elicited by these four Envs were compared to each other and to those elicited by a well-characterized clade B Env immunogen derived from the SF162 virus, which was isolated during chronic infection. Only one clade A Env, the one with the fewer glycosylation sites, elicited homologous neutralizing antibodies (NAbs); these did not target the V1, V2, or V3 regions. In contrast, all four clade A Envs elicited anti-V3 NAbs against "easy-to-neutralize" clade B and clade A isolates, irrespective of the variable region length and extent of glycosylation of the Env used as an immunogen. These anti-V3 NAbs did not access their epitopes on homologous and heterologous clade A, or B, neutralization-resistant viruses. The length and extent of glycosylation of the variable regions on the clade A Env immunogens tested did not affect the breadth of the elicited NAbs. Our data also indicate that the development of cross-reactive NAbs against clade A viruses faces similar hurdles to the development of cross-reactive anti-clade B NAbs.

Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization

Efficient in vitro systems to study the life cycle of hepatitis C virus (HCV) were recently developed for JFH1 (genotype 2a), which has unique replication capacity in Huh7 cells. We developed 4a/JFH1 intergenotypic recombinants containing the structural genes (Core, E1, and E2), p7, and all or part of NS2 of the 4a prototype strain ED43 that, after transfection of Huh7.5 cells with RNA transcripts, produced infectious viruses. Compared with the J6/JFH control virus, production of viruses was delayed. However, efficient spread of infection and high HCV RNA and infectivity titers were obtained in serial passages. Sequence analysis of recovered viruses and subsequent reverse genetic studies revealed a vital dependence on one or two NS2 mutations, depending on the 4a/2a junction. Infectivity of ED43/JFH1 viruses was CD81 dependent. The genotype 4 cell culture systems permit functional analyses as well as drug and vaccine research on an increasingly important genotype in the Middle East, Africa, and Europe. We also developed genotype 1a intergenotypic recombinants from H77C with vital mutations in NS3. Using H77C/JFH1 and ED43/JFH1 viruses, we demonstrated high homologous neutralizing antibody titers in 1a and 4a patient sera, respectively. Furthermore, availability of JFH1 viruses with envelope proteins of the six major HCV genotypes permitted cross-neutralization studies; 1a and 4a serum cross-neutralized 1a, 4a, 5a, and 6a but not 2a and 3a viruses. Thus, the JFH1 intergenotypic recombinants will be of importance for future studies of HCV neutralization and accelerate the development of passive and active immunoprophylaxis.