A potent broadly neutralizing human RSV antibody targets conserved site IV of the fusion glycoprotein.

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.

Potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopes.

There is still no safe and effective vaccine against dengue virus infection. Epidemics of dengue virus infection are increasingly a threat to human health around the world. Antibodies generated in response to dengue infection have been shown to impact disease development and effectiveness of dengue vaccine. In this study, we investigated monoclonal antibody responses to an experimental dengue vaccine in rhesus macaques. Variable regions of both heavy chain (VH) and light chain (VL) were cloned from single antibody-secreting B cells. A total of 780 monoclonal antibodies (mAbs) composed of paired VH and VL were characterized. Results show that the vaccination induces mAbs with diverse germline sequences and a wide range of binding affinities. Six potent neutralizing mAbs were identified among 130 dengue envelope protein binders. Critical amino acids for each neutralizing antibody binding to the dengue envelope protein were identified by alanine scanning of mutant libraries. Diverse epitopes were identified, including epitopes on the lateral ridge of DIII, the I-III hinge, the bc loop adjacent to the fusion loop of DII, and the ß-strands and loops of DI. Significantly, one of the neutralizing mAbs has a previously unknown epitope in DII at the interface of the envelope and membrane protein and is capable of neutralizing all four dengue serotypes. Taken together, the results of this study not only provide preclinical validation for the tested experimental vaccine, but also shed light on a potential application of the rhesus macaque model for better dengue vaccine evaluation and design of vaccines and immunization strategies.

Co-Administration of Lipid Nanoparticles and Sub-Unit Vaccine Antigens Is Required for Increase in Antigen-Specific Immune Responses in Mice.

A vast body of evidence suggests that nanoparticles function as potent immune-modulatory agents. We have previously shown that Merck proprietary Lipid NanoParticles (LNPs) markedly boost B-cell and T-cell responses to sub-unit vaccine antigens in mice. To further evaluate the specifics of vaccine delivery and dosing regimens in vivo, we performed immunogenicity studies in BALB/c and C57BL/6 mice using two model antigens, Hepatitis B Surface Antigen (HBsAg) and Ovalbumin (OVA), respectively. To assess the requirement for co-administration of antigen and LNP for the elicitation of immune responses, we evaluated immune responses after administering antigen and LNP to separate limbs, or administering antigen and LNP to the same limb but separated by 24 h. We also evaluated formulations combining antigen, LNP, and aluminum-based adjuvant amorphous aluminum hydroxylphosphate sulfate (MAA) to look for synergistic adjuvant effects. Analyses of antigen-specific B-cell and T-cell responses from immunized mice revealed that the LNPs and antigens must be co-administered-both at the same time and in the same location-in order to boost antigen-specific immune responses. Mixing of antigen with MAA prior to formulation with LNP did not impact the generation of antigen-specific B-cell responses, but drastically reduced the ability of LNPs to boost antigen-specific T-cell responses. Overall, our data demonstrate that the administration of LNPs and vaccine antigen together enables their immune-stimulatory properties.

IMPre: An Accurate and Efficient Software for Prediction of T- and B-Cell Receptor Germline Genes and Alleles from Rearranged Repertoire Data.

Large-scale study of the properties of T-cell receptor (TCR) and B-cell receptor (BCR) repertoires through next-generation sequencing is providing excellent insights into the understanding of adaptive immune responses. Variable(Diversity)Joining [V(D)J] germline genes and alleles must be characterized in detail to facilitate repertoire analyses. However, most species do not have well-characterized TCR/BCR germline genes because of their high homology. Also, more germline alleles are required for humans and other species, which limits the capacity for studying immune repertoires. Herein, we developed "Immune Germline Prediction" (IMPre), a tool for predicting germline V/J genes and alleles using deep-sequencing data derived from TCR/BCR repertoires. We developed a new algorithm, "Seed_Clust," for clustering, produced a multiway tree for assembly and optimized the sequence according to the characteristics of rearrangement. We trained IMPre on human samples of T-cell receptor beta (TRB) and immunoglobulin heavy chain and then tested it on additional human samples. Accuracy of 97.7, 100, 92.9, and 100% was obtained for TRBV, TRBJ, IGHV, and IGHJ, respectively. Analyses of subsampling performance for these samples showed IMPre to be robust using different data quantities. Subsequently, IMPre was tested on samples from rhesus monkeys and human long sequences: the highly accurate results demonstrated IMPre to be stable with animal and multiple data types. With rapid accumulation of high-throughput sequence data for TCR and BCR repertoires, IMPre can be applied broadly for obtaining novel genes and a large number of novel alleles. IMPre is available at https://github.com/zhangwei2015/IMPre.

A replication-defective human cytomegalovirus vaccine for prevention of congenital infection.

Congenital human cytomegalovirus (HCMV) infection occurs in ~0.64% of infants born each year in the United States and is the leading nongenetic cause of childhood neurodevelopmental disabilities. No licensed HCMV vaccine is currently available. Natural immunity to HCMV in women before pregnancy is associated with a reduced risk of fetal infection, suggesting that a vaccine is feasible if it can reproduce immune responses elicited by natural infection. On the basis of this premise, we developed a whole-virus vaccine candidate from the live attenuated AD169 strain, with genetic modifications to improve its immunogenicity and attenuation. We first restored the expression of the pentameric gH/gL/pUL128-131 protein complex, a major target for neutralizing antibodies in natural immunity. We then incorporated a chemically controlled protein stabilization switch in the virus, enabling us to regulate viral replication with a synthetic compound named Shield-1. The virus replicated as efficiently as its parental virus in the presence of Shield-1 but failed to produce progeny upon removal of the compound. The vaccine was immunogenic in multiple animal species and induced durable neutralizing antibodies, as well as CD4+ and CD8+ T cells, to multiple viral antigens in nonhuman primates.

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates.

Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.

Characterization of N-glycosylation profiles from mammalian and insect cell derived chikungunya VLP.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe arthralgia. The envelope of CHIKV is composed of 240 copies of two glycoproteins: E1 and E2. In this work, we have characterized the N-glycosylation patterns of CHIKV virus-like particles (VLPs), containing both E1 and E2 proteins, derived from mammalian and insect cells using hydrophilic interaction liquid chromatography (HILIC) with fluorescence (FL) and mass spectrometry (MS) detection. While HEK293 derived CHIKV VLPs contain oligomannose, hybrid and complex glycans, VLPs derived from SfBasic predominantly contain oligomannose glycans. This strong host dependence of N-glycosylation pattern resembles other alphaviruses such as SINV. The VLPs from HEK293 and SfBasic, with significantly different N-glycosylation profiles, are valuable reagents enabling future in-depth correlation studies between immunogenicity and glycosylation. In addition, the characterization tools presented here allow one to monitor glycosylation during vaccine process development and ensure process consistency.

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.

Pre-vaccination inflammation and B-cell signalling predict age-related hyporesponse to hepatitis B vaccination.

Aging is associated with hyporesponse to vaccination, whose mechanisms remain unclear. In this study hepatitis B virus (HBV)-naive older adults received three vaccines, including one against HBV. Here we show, using transcriptional and cytometric profiling of whole blood collected before vaccination, that heightened expression of genes that augment B-cell responses and higher memory B-cell frequencies correlate with stronger responses to HBV vaccine. In contrast, higher levels of inflammatory response transcripts and increased frequencies of pro-inflammatory innate cells correlate with weaker responses to this vaccine. Increased numbers of erythrocytes and the haem-induced response also correlate with poor response to the HBV vaccine. A transcriptomics-based pre-vaccination predictor of response to HBV vaccine is built and validated in distinct sets of older adults. This moderately accurate (area under the curve≈65%) but robust signature is supported by flow cytometry and cytokine profiling. This study is the first that identifies baseline predictors and mechanisms of response to the HBV vaccine.

A novel lipid nanoparticle adjuvant significantly enhances B cell and T cell responses to sub-unit vaccine antigens.

Sub-unit vaccines are primarily designed to include antigens required to elicit protective immune responses and to be safer than whole-inactivated or live-attenuated vaccines. But their purity and inability to self-adjuvant often result in weaker immunogenicity. Emerging evidence suggests that bio-engineered nanoparticles can be used as immunomodulatory adjuvants. Therefore, in this study we explored the potential of novel Merck-proprietary lipid nanoparticle (LNP) formulations to enhance immune responses to sub-unit viral antigens. Immunization of BALB/c and C57BL/6 mice revealed that LNPs alone or in combination with a synthetic TLR9 agonist, immune-modulatory oligonucleotides, IMO-2125 (IMO), significantly enhanced immune responses to hepatitis B virus surface antigen (HBsAg) and ovalbumin (OVA). LNPs enhanced total B-cell responses to both antigens tested, to levels comparable to known vaccine adjuvants including aluminum based adjuvant, IMO alone and a TLR4 agonist, 3-O-deactytaled monophosphoryl lipid A (MPL). Investigation of the quality of B-cell responses demonstrated that the combination of LNP with IMO agonist elicited a stronger Th1-type response (based on the IgG2a:IgG1 ratio) than levels achieved with IMO alone. Furthermore, the LNP adjuvant significantly enhanced antigen specific cell-mediated immune responses. In ELISPOT assays, depletion of specific subsets of T cells revealed that the LNPs elicited potent antigen-specific CD4(+) and CD8(+)T cell responses. Intracellular FACS analyses revealed that LNP and LNP+IMO formulated antigens led to higher frequency of antigen-specific IFNγ(+)TNFα(+)IL-2(+), multi-functional CD8(+)T cell responses, than unadjuvanted vaccine or vaccine with IMO only. Overall, our results demonstrate that lipid nanoparticles can serve as future sub-unit vaccine adjuvants to boost both B-cell and T-cell responses in vivo, and that addition of IMO can be used to manipulate the quality of immune responses.

Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures.

Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization.

Effector and Central Memory Poly-Functional CD4(+) and CD8(+) T Cells are Boosted upon ZOSTAVAX(®) Vaccination.

ZOSTAVAX(®) is a live attenuated varicella-zoster virus (VZV) vaccine that is licensed for the protection of individuals ≥50 years against shingles and its most common complication, postherpetic neuralgia. While IFNγ responses increase upon vaccination, the quality of the T cell response has not been elucidated. By using polychromatic flow cytometry, we characterized the breadth, magnitude, and quality of ex vivo CD4(+) and CD8(+) T cell responses induced 3-4 weeks after ZOSTAVAX vaccination of healthy adults. We show, for the first time that the highest frequencies of VZV-specific CD4(+) T cells were poly-functional CD154(+)IFNγ(+)IL-2(+)TNFα(+) cells, which were boosted upon vaccination. The CD4(+) T cells were broadly reactive to several VZV proteins, with immediate early (IE) 63 ranking the highest among them in the fold rise of poly-functional cells, followed by IE62, gB, open reading frame (ORF) 9, and gE. We identified a novel poly-functional ORF9-specific CD8(+) T cell population in 62% of the subjects, and these were boosted upon vaccination. Poly-functional CD4(+) and CD8(+) T cells produced significantly higher levels of IFNγ, IL-2, and TNFα compared to mono-functional cells. After vaccination, a boost in the expression of IFNγ by poly-functional IE63- and ORF9-specific CD4(+) T cells and IFNγ, IL-2, and TNFα by ORF9-specific poly-functional CD8(+) T cells was observed. Responding poly-functional T cells exhibited both effector (CCR7(-)CD45RA(-)CD45RO(+)), and central (CCR7(+)CD45RA(-)CD45RO(+)) memory phenotypes, which expressed comparable levels of cytokines. Altogether, our studies demonstrate that a boost in memory poly-functional CD4(+) T cells and ORF9-specific CD8(+) T cells may contribute toward ZOSTAVAX efficacy.

Preclinical and clinical development of a dengue recombinant subunit vaccine.

This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 µg and 50 µg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 µg DEN1-80E and the 50 µg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.

Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates.

We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIX™ adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 µg/dose formulated with ISCOMATRIX™ adjuvant, were equally immunogenic. A group immunized with 20 µg DEN4-80E and Alhydrogel™ induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 µg groups and all but one in the DEN4-80EZip 100 µg group were protected from viremia. Two out of three monkeys in the Alhydrogel™ group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 µg of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 µg) and high (50, 50, 50, 100 µg) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose.

DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge.

Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4(+) T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins.

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

Development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a Chikungunya virus-like particle vaccine.

To effectively support the development of a Chikungunya (CHIKV) virus-like particle (VLP) vaccine, a sensitive and robust high-performance liquid chromatography (HPLC) method that can quantitate CHIKV VLPs and monitor product purity throughout the manufacturing process is needed. We developed a sensitive reversed-phase HPLC (RP-HPLC) method that separates capsid, E1, and E2 proteins in CHIKV VLP vaccine with good resolution. Each protein component was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). The post-translational modifications on the viral glycoproteins E1 and E2 were further identified by intact protein mass measurements with liquid chromatography-mass spectrometry (LC-MS). The RP-HPLC method has a linear range of 0.51-12 µg protein, an accuracy of 96-106% and a precision of 12% RSD, suitable for vaccine product release testing. In addition, we demonstrated that the RP-HPLC method is useful for characterizing viral glycoprotein post-translational modifications, monitoring product purity during process development and assessing product stability during formulation development.

A herpes simplex virus 2 (HSV-2) glycoprotein D-expressing nonreplicating dominant-negative HSV-2 virus vaccine is superior to a gD2 subunit vaccine against HSV-2 genital infection in guinea pigs.

We recently constructed a novel non-replicating dominant-negative HSV-2 recombinant viral vaccine (CJ2-gD2) capable of expressing various HSV-2 antigens that are dominant targets of HSV-2-specific CD8 T-cell response. Importantly, CJ2-gD2 expresses gD2, the HSV-2 major antigen glycoprotein D, as efficiently as wild-type HSV-2 infection and can lead to a nearly 500-fold reduction in wild-type HSV-2 viral replication in cells co-infected with CJ2-gD2 and wild-type HSV-2. In this report, we show that CJ2-gD2 elicits a strong antibody response to various HSV-2 antigens and is highly effective in the prevention of primary and recurrent HSV-2 genital infection and disease in the immunized guinea pigs. The direct comparison study between CJ2-gD2 and a gD2 subunit vaccine (gD2-alum/MPL) with a formulation akin to a vaccine tested in phase III clinical trials shows that CJ2-gD2 is 8 times more effective than the gD2-alum/MPL subunit vaccine in eliciting an anti-HSV-2 specific neutralizing antibody response and offers significantly superior protection against primary and recurrent HSV-2 genital infections. Importantly, no challenge wild-type HSV-2 viral DNA was detectable in dorsal root ganglia DNA isolated from CJ2-gD2-immunized guinea pigs on day 60 post-challenge. CJ2-gD2 should be an excellent HSV-2 vaccine candidate for protection against HSV-2 genital infection and disease in humans.

Enhanced production of Chikungunya virus-like particles using a high-pH adapted spodoptera frugiperda insect cell line.

Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models and are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable and robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in standard culture media (pH 6.2-6.4), properly processed Chikungunya structural proteins were detected and assembled capsids were observed. However, an increase in culture pH to 6.6-6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability and Sf21 culture, medium modifications were made and a novel insect cell variant (SfBasic) was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0-7.2 and produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line and production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein and VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.

Protection provided by a herpes simplex virus 2 (HSV-2) glycoprotein C and D subunit antigen vaccine against genital HSV-2 infection in HSV-1-seropositive guinea pigs.

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.