Search

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed.

Dissolved carbon dioxide (dCO2 ) accumulation during cell culture has been recognized as an important parameter that needs to be controlled for successful scale-up of animal cell culture because above a certain concentration there are adverse effects on cell growth performance and protein production. We investigated the effect of accumulation of dCO2 in bioreactor cultures of expresSF+(®) insect cells infected with recombinant baculoviruses expressing recombinant influenza virus hemagglutinins (rHA). Different strategies for bioreactor cultures were used to obtain various ranges of concentrations of dCO2 ( 200 mmHg) and to determine their effects on recombinant protein production and cell metabolic activity. We show that the accumulation of dCO2 at levels > 100 mmHg resulted in reduced metabolic activity, slowed cell growth, prolonged culture viability after infection, and decreased infection kinetics. The reduced rHA yields were not caused by the decrease in the extracellular pH that resulted from dCO2 accumulation, but were most likely due to the effect of dCO2 accumulation in cells. The results obtained here at the 2 L scale have been used for the design of large-scale processes to manufacture the rHA based recombinant vaccine Flublok™ at the 2500 L scale Biotechnol. Bioeng. 2015;112: 2267-2275. © 2015 Wiley Periodicals, Inc.

Strategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens (Ag) for more long-lived, efficacious HIV-1 vaccine-induced B-cell responses. HIV-1 Env gp140 (native or uncleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells results in anergic effects on T-cell recruitment and consequently a lack of strong, robust, and durable B-memory responses. To test this hypothesis, we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3-kDa CD4 miniprotein mimetic, serving to block ligation of gp140 on CD4 + T cells while preserving CD4-inducible (CDi) neutralizing epitopes targeted by antibody-dependent cellular cytotoxicity (ADCC) effector responses. Importantly, immunization of rhesus macaques consistently gave superior B-cell ( P < 0.001) response kinetics and superior ADCC ( P < 0.014) in a group receiving the CD4bs-occluded vaccine compared to those of animals immunized with gp140. Of the cytokines examined, Ag-specific interleukin-4 (IL-4) T-helper enzyme-linked immunosorbent spot (ELISpot) assays of the CD4bs-occluded group increased earlier ( P = 0.025) during the inductive phase. Importantly, CD4bs-occluded gp140 antigen induced superior B-cell and ADCC responses, and the elevated B-cell responses proved to be remarkably durable, lasting more than 60 weeks postimmunization. IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have been unsuccessful until now. Antigen-specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T cells as soluble shed antigen or as antigen-antibody complexes, causing impairment in the activation of these specialized CD4-positive T cells. We proposed that CD4-binding impairment is partly responsible for the relatively poor B-cell responses to HIV envelope-based vaccines. To test this hypothesis, we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.

Evaluation of the epitope specificities, locations (systemic or mucosal), and effector functions of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitudes, epitope specificities, avidities, and functions of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4-mimetic miniprotein (gp140-M64U1) in rhesus macaques. Cross-linking of gp140 Env to M64U1 resulted in earlier increases of both the magnitude and avidity of the IgG binding response than those with Env protein alone. Notably, IgG binding responses at an early time point correlated with antibody-dependent cellular cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the cross-linked Env group than for the Env group. In addition, the cross-linked Env group developed higher IgG responses against a linear epitope in the gp120 C1 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by cross-linking of gp140 with the CD4-mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses, correlating with the rise of subsequent antibody-mediated antiviral functions. IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and immune correlate analysis of that trial revealed a role for binding antibodies and antibody Fc-mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope, with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen designs in nonhuman primates is critical for understanding how to improve upon immunogen design to inform further testing in human clinical trials. Our results demonstrate that structural modifications of Env that aim to mimic the CD4-bound conformation can result in earlier antibody elicitation, altered epitope specificity, and increased antiviral function postimmunization.

Multiple different hemagglutinin (HA) protein antigens have been reproducibly manufactured at the 650L scale by Protein Sciences Corporation (PSC) based on an insect cell culture with baculovirus infection. Significantly, these HA protein antigens were produced by the same Universal Manufacturing process as described in the biological license application (BLA) for the first recombinant influenza vaccine approved by the FDA (Flublok). The technology is uniquely designed so that a change in vaccine composition can be readily accommodated from one HA protein antigen to another one. Here we present a vaccine candidate to combat the recently emerged H7N9 virus as an example starting with the genetic sequence for the required HA, creation of the baculovirus and ending with purified protein antigen (or vaccine component) at the 10L scale accomplished within 38 days under GMP conditions. The same process performance is being achieved at the 2L, 10L, 100L, 650L and 2500L scale. An illustration is given of how the technology was transferred from the benchmark 650L scale facility to a retrofitted microbial facility at the 2500L scale within 100 days which includes the time for facility engineering changes. The successful development, technology transfer and scale-up of the Flublok process has major implications for being ready to make vaccine rapidly on a worldwide scale as a defense against pandemic influenza. The technology described does not have the same vulnerability to mutations in the egg adapted strain, and resulting loss in vaccine efficacy, faced by egg based manufacture.

Chikungunya virus (CHIKV) is an alphavirus that infects millions of people every year, especially in the developing world. The selective expression of recombinant CHIKV capsid and envelope proteins results in the formation of self-assembled virus-like particles (VLPs) that have been shown to protect nonhuman primates against infection from multiple strains of CHIKV. This study describes the characterization, excipient screening, and optimization of CHIKV VLP solution conditions toward the development of a stable parenteral formulation. The CHIKV VLPs were found to be poorly soluble at pH 6 and below. Circular dichroism, intrinsic fluorescence, and static and dynamic light scattering measurements were therefore performed at neutral pH, and results consistent with the formation of molten globule structures were observed at elevated temperatures. A library of generally recognized as safe excipients was screened for their ability to physically stabilize CHIKV VLPs using a high-throughput turbidity-based assay. Sugars, sugar alcohols, and polyanions were identified as potential stabilizers and the concentrations and combinations of select excipients were optimized. The effects of polyanions were further studied, and while all polyanions tested stabilized CHIKV VLPs against aggregation, the effects of polyanions on conformational stability varied.

Objective: To study the complex formed between Tat protein and Env soluble trimeric immunogen, and compare with previously determined structures of Env native trimers and Env–CD4m complexes. Design: The soluble Env trimer was used to mimic the spike glycoprotein on the virus surface for the study. To overcome limitations of other structural determination methods, cryoelectron microscopy was employed to image the complex, and single particle reconstruction was utilized to reconstruct the structure of the complex from collected micrographs. Molecular modeling of gp120–Tat was performed to provide atomic coordinates for docking. Methods: Images were preprocessed by multivariate statistical analysis to identify principal components of variation then submitted for reconstruction. Reconstructed structures were docked with modeled gp120–Tat atomic coordinates to study the positions of crucial epitopes. Results: Analysis of the Env–Tat complex demonstrated an intermediate structure between Env native trimers and Env–CD4m structures. Docking results indicate that the CD4-binding site and the V3 loop are exposed in the Env–Tat complex. The integrin-binding sequence in Tat was also exposed in Env–Tat docking. Conclusion: The intermediate structure induced by Tat-interaction with Env could potentially provide an explanation for increased virus infection in the presence of Tat protein. Consequently, exposure of CD4-binding sites and a putative integrin-binding sequence on Tat in the complex may provide a new avenue for rational design of an effective HIV vaccine.

Previously, priming rhesus macaques with Adenovirus type 5 host range mutant-recombinants encoding Tat and Env and boosting with Tat and Env protein in MPL-SE controlled chronic viremia by 4 logs following homologous intravenous SHIV89.6P challenge. Here we evaluated Tat, Env, and Tat/Env regimens for immunogenicity and protective efficacy using clade C Env, alum adjuvant, and a heterologous intrarectal SHIV1157ipd3N4 challenge. Despite induction of strong cellular and humoral immunity, Tat/Env group T and B-cell memory responses were not significantly enhanced over Tat- or Env-only groups. Lack of viremia control post-challenge was attributed to lower avidity Env antibodies and no anamnestic ADCC response or SHIV1157ipd3N4 neutralizing antibody development post-challenge. Poor biologic activity of the Tat immunogen may have impaired Tat immunity. In the absence of sterilizing immunity, strong anamnestic responses to heterologous virus can help control viremia. Both antibody breadth and optimal adjuvanticity are needed to elicit high-quality antibody for protective efficacy.

The importance of the strength of antigen adsorption by aluminum-containing adjuvants on immunopotentiation was studied using HIV 1 SF162dV2gp140 (gp140), a potential HIV/AIDS antigen. The strengths of adsorption by aluminum hydroxide (AH) adjuvant and aluminum phosphate adjuvant, as measured by the Langmuir adsorptive coefficient, were 1900 and 400 mL/mg, respectively. The strength of adsorption by AH was modified by pretreatment of AH with two different concentrations of potassium dihydrogen phosphate to produce phosphate-treated aluminum hydroxide adjuvants having adsorptive coefficients of 1200 and 800 mL/mg. The four adjuvants were used to prepare vaccines containing either 1 or 10 μg of gp140 per dose. Antibody studies in mice revealed that the presence of an adjuvant increased the immune response in comparison with a solution of gp140 when the dose was 1 μg. Furthermore, the immune response was inversely related to the adsorptive coefficient. In contrast, no significant difference in immunopotentiation was observed between treatments in the presence or absence of an adjuvant when the dose of gp140 was 10 μg. Analysis of the binding of gp140 to CD4 and anti-gp140 monoclonal antibodies by surface plasmon resonance suggests that tight binding induced structural changes in the antigen.

A major goal of human immunodeficiency virus type 1 (HIV-1) vaccine efforts is the design of Envelope (Env)-based immunogens effective at eliciting heterologous or broad neutralizing antibodies (NAbs). We hypothesized that programming the B-cell response could be achieved by sequentially exposing the host to a collection of env variants representing the viral quasispecies members isolated from an individual that developed broad NAbs over time. This ordered vaccine approach (sequential) was compared to exposure to a cocktail of env clones (mixture) and to a single env variant (clonal). The three strategies induced comparable levels of the autologous and heterologous neutralization of tier 1 pseudoviruses. Sequential and mixture exposure to quasispecies led to epitope targeting similar to that observed in the simian-human immunodeficiency virus (SHIV)-infected animal from which the env variants were cloned, while clonal and sequential exposure led to greater antibody maturation than the mixture. Therefore, the sequential vaccine approach best replicated the features of the NAb response observed in that animal. This study is the first to explore the use of a collection of HIV-1 env quasispecies variants as immunogens and to present evidence that it is possible to educate the B-cell response by sequential exposure to native HIV-1 quasispecies env variants derived from an individual with a broadened NAb response.

HIV native Tat and V2 loop-deleted Env (EnvΔV2) proteins already proved safe and immunogenic in phase I clinical testing as single vaccine components. Further, a phase II vaccine trial with Tat showed intensification of the therapeutic effects of HAART in successfully treated HIV-infected individuals. Here a pilot study assessed the immunogenicity and protective efficacy of an HIV/AIDS vaccine based on the combination of Tat and EnvΔV2 proteins in cynomolgus macaques against homologous intrarectal challenge with 35 MID(50) (monkey infectious dose 50) of an R5 simian-human immunodeficiency virus (SHIV(SF162P4cy)). Upon challenge, three of four macaques immunized with Tat and EnvΔV2, and two of three monkeys immunized with EnvΔV2 alone were protected from infection. In contrast, all three control animals, which had been either administered with the adjuvants only or left untreated, and an additional monkey immunized with Tat alone became systemically infected. Protection of the macaques vaccinated with EnvΔV2 or Tat/EnvΔV2 correlated with higher peak titers of pre-challenge neutralizing antibodies obtained during the immunization period (between 70 and 3 weeks before challenge) and with anti-Env V3 loop binding antibodies assessed 3 weeks before challenge. Compared to EnvΔV2 alone, the Tat and EnvΔV2 combined vaccine elicited faster antibody responses (IgM) with a trend, early in the vaccination schedule, after the second immunization including EnvΔV2, towards broader anti-Env IgG epitope specificity and a higher ratio of neutralizing to Env-binding antibody titers. As the number of immunizations increased, vaccination with EnvΔV2 approached the immune response assessed after two inocula with the Tat/EnvΔV2 combined vaccine, even though some differences remained between groups, as indicated by anti-Env IgG epitope mapping. In fact, three weeks before challenge, plasma IgG of animals in the EnvΔV2 group showed a trend towards stronger specificity for the V1 loop and V5 loop-C5 regions of Env, whereas the Tat/EnvΔV2 group displayed an overall higher reactivity for epitopes within the Env V3 loop throughout the immunization period. Although differences in terms of protection rate were not found between the EnvΔV2 or Tat/EnvΔV2 vaccination groups in this pilot study, vaccination with Tat/EnvΔV2 appeared to accelerate the induction of potentially protective antibody responses to Env. In particular, antibodies to the Env V3 loop, whose levels at pre-challenge correlated with protection, were already higher early in the vaccination schedule in monkeys immunized with Tat/EnvΔV2 as compared to EnvΔV2 alone. Further studies including larger vaccination groups and fewer immunizations with these two vaccine candidates are needed to confirm these findings and to assess whether the Tat/EnvΔV2 vaccine may afford superior protection against infection.

Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.

Improving the potency, breadth, and durability of neutralizing antibody responses to HIV are major challenges for HIV vaccine development. To address these challenges, the studies described evaluate in rabbits the titers, breadth, and epitope specificities of antibody responses elicited by HIV envelope subunit vaccines adjuvanted with MF59 with or without CpG oligodeoxynucleotide (ODN). Animals were immunized with trimeric o-gp140ΔV2 derived from subtype B HIV-1SF162 or subtype C HIV-1TV1, or proteins from both strains. Immunization with SF162 or TV1 with MF59/CpG elicited higher titers of binding and neutralizing antibodies to SF162 than monovalent immunization with MF59 alone (P

International audience; To counteract the problems associated with the purification of HIV envelope, we developed a new purification method exploiting the high affinity of a peptide mimicking CD4 towards the viral glycoprotein. This miniCD4 was used as a ligand in affinity chromatography and allowed the separation in one step of HIV envelope monomer from cell supernatant and the capture of pre-purified trimer. This simple and robust method of purification yielded to active and intact HIV envelopes as proved by the binding of CCR5 HIV co-receptor, CD4 and a panel of well-characterized monoclonal antibodies. The immunogenicity of miniCD4-purified HIV envelope was further assessed in rats. The analysis of the humoral response indicated that elicited antibodies were able to recognize a broad range of HIV envelopes. Finally, this method based on a chemically synthesized peptide may represent a convenient and versatile tool for protein purification compatible far scale-up in both academic and pharmaceutical researches.

CD40-CD154 interaction forms a key event in regulation of crosstalk between dendritic cells and CD4 T cells. In human immunodeficiency virus (HIV)-1 infected patients CD154 expression is impaired, and the resulting loss of immune responsiveness by CD4+ T cells contributes to a progressive state of immunodeficiency in humans. Although chimpanzees are susceptible to chronic HIV-1/SIVcpz infection, they are relatively resistant to the onset of AIDS. This relative resistance is characterized by maintenance of CD4+ T cell populations and function, which is highly compromised in human patients. In our cohort of chronically HIV-1- and SIVcpz-infected chimpanzees, we demonstrated the capacity to produce IL-2, following CD3/CD28 stimulation, as well as preserved CD154 up-regulation. Cross-linking of CD4 with mAb was found to inhibit CD3/CD28-induced up-regulation of CD154 equally in chimpanzees and humans. However, specific cross-linking with trimeric recombinant HIV-1 gp140 revealed reduced sensitivity for inhibition of CD154 up-regulation in chimpanzees, requiring fourfold higher concentrations of viral protein. Chimpanzee CD4+ T cells are thus less sensitive to the immune-suppressive effect of low-dose HIV-1 envelope protein than human CD4+ T cells.

Background: Worldwide, the majority of human immunodeficiency virus (HIV) infections occur by heterosexual transmission. Thus, the development of a vaccine that can prevent intravaginal HIV infection is an important goal of AIDS vaccine research. Objectives: To determine which single or combination of systemic and mucosal routes of immunizations of female rhesus macaques with an HIV-1SF162 envelope protein vaccine induced protection against intravaginal challenge with SHIV. Design: Female rhesus macaques were immunized with an HIV-1SF162 envelope protein vaccine administered systemically (intramuscularly), or mucosally (intranasally), or as a sequential combination of both routes. The macaques were then challenged intravaginally with SHIVSF162P4, expressing an envelope that is closely matched (homologous) to the vaccine. Results: Macaques receiving intramuscular immunizations, alone or in combination with intranasal immunizations, were protected from infection, with no detectable plasma viral RNA, provirus, or seroconversion to nonvaccine viral proteins, and better preservation of intestinal CD4þ T cells. Serum neutralizing antibodies against the challenge virus appeared to correlate with protection. Conclusions: The results of this study demonstrate that, in the nonhuman primate model, it is possible for vaccine-elicited immune responses to prevent infection after intravaginal administration of virus. 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins AIDS 2008, 22:339‐348

We have previously shown that the biologically active Tat protein targets and efficiently enters dendritic cells, and increases the proteolytic activities of the immunoproteasome, thereby favoring the generation and presentation of the subdominant MHC-I binding CTL epitopes of heterologous antigens. In the present study, we demonstrate that Tat broadens in vivo epitope-specific T cell responses directed to heterologous antigens including HIV structural proteins. Specifically, co-immunization of mice with OVA and Tat proteins induces CTL responses against subdominant and cryptic OVA-derived epitopes, which are not detected in mice vaccinated with OVA alone. Similarly, mice vaccinated with the HIV-1 Gag, Env or V2-deleted Env antigens in combination with Tat show Th1-type and CTL responses directed to a larger number of T cell epitopes, as compared to mice vaccinated with these proteins in absence of Tat. In contrast, Tat did not affect Th2-type responses to these structural HIV proteins. These results indicate that Tat is not only an antigen but also a novel Th1-type adjuvant capable of broadening in vivo the spectrum of epitopes recognized by T cells, and suggest that Tat can be considered an optimal co-antigen in the development of novel vaccination strategies against AIDS.

Pre-clinical HIV-1 vaccine protocols, using multiple vaccine modalities and a potent adjuvant were assessed for vaccine efficacy in an experimental HIV-1 challenge model. C57Bl/6 mice were immunized with DNA plasmids encoding HIV-1 gp140, Gag and Tat alone or in combination with the corresponding recombinant proteins formulated in the adjuvant MF59. HIV-1 DNA alone or a DNA prime protein boost schedule resulted in complete protection against challenge with HIV-1/MuLV-infected murine cells. Although HIV-1 protein immunization in combination with MF59 resulted in partial protection, the DNA priming seemed to be crucial for obtaining full protection against the challenge. It is likely that the partial protection seen after immunization with protein alone is, to a certain extent, due to effects of the adjuvant since some animals that received the adjuvant MF59 alone were protected from the challenge. For the most part, antigen-specific cell-mediated immune responses as detected in the spleen (in contrast to responses detected in peripheral blood) of immunized animals appeared to be associated with protection in this study.

Eleven anti-HIV Env monoclonal antibodies (MAbs) were isolated from mice immunized with soluble Env proteins derived from the clade B Env, SF162, or ΔV2 (a derivative of SF162 lacking the V2 loop). All six anti-gp120 MAbs studied, neutralized SF162 and their activities were dependent by the glycosylation patterns of the V1, V2 or V3 loops. Only one anti-gp120 MAb (an anti-V3 MAb) displayed cross-neutralizing activity, which was influenced by the type of V1 loop present on the target heterologous viruses. None of the five anti-gp41 MAbs studied displayed anti-SF162 neutralizing activity. Our studies indicate that the current limitation of soluble HIV Env gp140 immunogens to elicit robust cross-reactive neutralizing antibody responses is not only due to the elicitation of high titers of homologous antibodies but also due to the elicitation of antibodies whose epitopes are naturally occluded, or not present, on the virion-associated Env.

With more than 40 million people living with human immunodeficiency virus (HIV), there is an urgent need to develop drugs that can be used in the form of a topical microbicide to prevent infection through sexual transmission. DCM205 is a recently discovered small-molecule inhibitor of HIV type 1 (HIV-1) that is able to directly inactivate HIV-1 in the absence of a cellular target. DCM205 is active against CXCR4-, CCR5-, and dual-tropic laboratory-adapted and primary strains of HIV-1. DCM205 binds to the HIV-1 envelope glycoprotein, and competition studies map the DCM205 binding at or near the V3 loop of gp120. Binding to this site interferes with the soluble CD4 interaction. With its ability to disable the virus particle, DCM205 represents a promising new class of HIV entry inhibitor that can be used as a strategy in the prevention of HIV-1/AIDS.

This study was designed to improve the stability of liquid formulations of recombinant influenza hemagglutinin (rHA) and to understand the mechanism of early loss of potency for rHA. The potency of rHA derived from several influenza strains was determined using single radial immunodiffusion (SRID), and the structure of the rHA was characterized using SDS-PAGE and dynamic light scattering. rHA formed disulfide cross-linked multimers, and potency decreased during extended storage. To reduce disulfide-mediated cross-linking and early potency loss, rHA was formulated with sodium thioglycolate (STG) and citrate. Addition of 80 mM STG and 55 mM sodium citrate inhibited disulfide-mediated cross-linking without affecting protein function for each rHA tested. The shelf life of the rHA formulation with STG-citrate, based on potency as determined by SRID, was extended as much as 20-fold, compared to a control formulation without STG-citrate. STG-citrate did not have a significant effect on the immunogenicity of H1 A/California/7/2009 rHA in mice.

The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.

Although HIV subtype B predominates in North America and Western Europe, most HIV infections worldwide are non-subtype B. Globally effective AIDS vaccines need to elicit broad immunity against multiple HIV strains. In this study, 10 chimpanzees were intranasally primed sequentially with adenovirus type 5 (Ad5)-and Ad7-HIV MN env/rev recombinants and boosted twice intramuscularly with heterologous oligomeric HIV SF162 gpl40AV2 protein in MF59 adjuvant. Sera were evaluated for binding, neutralizing, and antibody-dependent cellular cytotoxicity (ADCC) against HIV clades A, B, C, and CRF01_AE. The vaccine regimen elicited high-titered HIV subtype A, B, C and CRF01_AE gp120-binding antibodies. Sera from 7 of 10 vaccinated chimpanzees cross-neutralized the heterologous South African subtype C primary HIV TV-1 isolate. Significant cross-clade neutralization against other subtype A, C and E isolates was not observed. Sera from all animals mediated ADCC of cells coated with gp120 from HIV subtypes A and B. Nine of 10 animals also exhibited ADCC activity against HIV subtype C and CRF01_AE gp120-coated targets. This subtype B Ad-HIV recombinant prime/envelope protein boost regimen is a promising approach for eliciting broad ADCC activity against diverse HIV clades. Incorporating additional non-subtype B envelope genes and protein boosts in a multivalent strategy may be required to elicit broader neutralizing antibodies against non-subtype B HIV strains.

The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIVSF162P4, and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, ΔV2gp140 (lacking the crown of the V2 loop), ΔV3gp140 (lacking the crown of the V3 loop), and ΔV2ΔV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and ΔV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and ΔV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by ΔV3gp140 or ΔV2ΔV3gp140. In contrast, the SHIVSF162P4-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.

Background Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form. Results The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25°C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach. Conclusion rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.

Hypervariable loops of HIV-1 Env protein gp120 are speculated to play roles in the conformational transition of Env to the receptor binding-induced metastable state. Structural analysis of full-length Env-based immunogens, containing the entire V2 loop, displayed tighter association between gp120 subunits, resulting in a smaller trimeric diameter than constructs lacking V2. A prominent basal quaternary location of V2 and V3′ that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops. Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location. The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports.

Dissolved carbon dioxide (dCO2 ) accumulation during cell culture has been recognized as an important parameter that needs to be controlled for successful scale-up of animal cell culture because above a certain concentration there are adverse effects on cell growth performance and protein production. We investigated the effect of accumulation of dCO2 in bioreactor cultures of expresSF+(®) insect cells infected with recombinant baculoviruses expressing recombinant influenza virus hemagglutinins (rHA). Different strategies for bioreactor cultures were used to obtain various ranges of concentrations of dCO2 (50, 50-100, 100-200, and200 mmHg) and to determine their effects on recombinant protein production and cell metabolic activity. We show that the accumulation of dCO2 at levels 100 mmHg resulted in reduced metabolic activity, slowed cell growth, prolonged culture viability after infection, and decreased infection kinetics. The reduced rHA yields were not caused by the decrease in the extracellular pH that resulted from dCO2 accumulation, but were most likely due to the effect of dCO2 accumulation in cells. The results obtained here at the 2 L scale have been used for the design of large-scale processes to manufacture the rHA based recombinant vaccine Flublok™ at the 2500 L scale Biotechnol. Bioeng. 2015;112: 2267-2275. © 2015 Wiley Periodicals, Inc.

The overall immune responses elicited naturally by human immunodeficiency virus (HIV) infection are not effective at controlling viral replication or disease progression. HIV establishes persistence by immune evasion strategies (13), inherently resisting neutralizing antibodies, repeatedly selecting mutants that escape antibody and T-cell immune responses, and avoiding cytotoxic T-lymphocyte (CTL) killing and impairing CD4 T-cell function by downregulation of major histocompatibility complex class I and CD4 molecules from the surface of infected cells. Since HIV is exquisitely adapted for pathogenesis, an efficacious HIV vaccine will need to induce broader, more potent cellular and humoral immune responses than those elicited by natural infection (7). Live viral vectors, such as adenovirus (Ad), as vaccine vehicles present one option for inducing more potent immunity. Ads are advantageous because they target epithelial cells of the upper respiratory tract and gut, inducing mucosal immunity critical for preventing HIV infection at genital and/or rectal sites. Ads infect immature dendritic cells (DC), leading to DC maturation and efficient antigen presentation of inserted viral gene products (49, 50). Ads are highly immunogenic and engage both arms of the immune system, eliciting long-lasting cellular and humoral immunity to inserted gene products. Replication-competent (replicating) Ad-HIV recombinants exploit the potential of Ad vectors for eliciting persistent immune responses. In replicating Ad recombinants, expression of the encoded HIV antigen is incorporated into the Ad replication cycle, so lower immunization doses can achieve longer and higher expression levels of HIV gene product in vivo than replication-defective Ad recombinants. In vivo replication of Ad recombinants stimulates production of proinflammatory cytokines that can augment immune responses. Apoptotic cells arising from Ad replication can provide DC with exogenous antigens for initiation of T-cell responses through cross-presentation (12). Although vaccine vectors may compete with transgenes for induction of immune responses (see below), strong immune responses to Ad antigens may paradoxically enhance immunity to transgene-encoded HIV antigens via CD8-T-cell-mediated autocrine help (39), whereby CD8+ T cells can provide help for other responding CD8+ T cells if present in sufficient numbers (43). A combination vaccine regimen involving priming with replicating Ad-HIV or simian immunodeficiency virus (SIV) recombinants and boosting with HIV or SIV envelope proteins has elicited strong cellular, humoral, and mucosal immune responses to inserted HIV and SIV gene products in both chimpanzee and macaque models (22, 23, 32, 51). Chimpanzees immunized with this regimen exhibited long-lasting protection against HIV challenges (22, 34), whereas macaques have shown significant protection against a highly pathogenic SIVmac251 challenge (33, 48). Recently, such priming with multigenic Ad-SIV recombinants and boosting with envelope protein subunits induced potent protection against SIVmac251 intrarectal challenge. A total of 39% of immunized macaques remained aviremic after challenge or cleared or controlled plasma viremia to the threshold of detection (33). Protection during the chronic phase of infection was correlated with vaccine-induced cellular immunity and during the acute phase of infection with anti-envelope binding antibodies. The latter antibodies have been recently shown to mediate antibody-dependent cellular cytotoxicity (ADCC), and the activity was significantly correlated with reduced acute-phase viremia (15). Replication-defective (nonreplicating) Ad recombinants lacking E1 genes required for replication are also being developed (6, 11, 20, 35). In macaques, a nonreplicating Ad5-SIVgag recombinant combined with SIVgag DNA priming very effectively induced high frequencies of SIV-specific T cells and significantly reduced viral burden after a SHIV89.6P challenge (35). Although these animal model results are encouraging, some obstacles need to be overcome before using Ad vaccines in humans. Preexisting immunity to Ad vectors can impede induction of effective immunity to encoded immunogens. Anti-Ad5 immunity has suppressed nonreplicating Ad/HIV vaccines in mice (4, 38, 46) and rhesus macaques (6). Since Ad-neutralizing antibodies are serotype specific, anti-Ad immunity may be surmounted to a large extent by use of alternative Ad vectors not prevalent in humans (10, 42) or by sequential immunization with Ad vectors of different serotype (22). Use of high-dose nonreplicating Ad vaccines (>1010 PFU) to overcome prior immunity, however, can result in significant toxicity and immunopathology (21, 28, 41). Lower doses of replication-defective Ad vector may avoid toxicity, but at the expense of an adequate immune response as seen in a phase I trial in which only 40% of the volunteers exhibited a positive response to the vaccine (8). Thus, circumventing anti-Ad immunity while using an immunization dose that avoids immunopathology and yet achieves sufficient antigen expression for induction of potent immune responses is critical. We hypothesized that sequential immunization with low doses of replicating Ad recombinants based in different serotypes would better induce potent persistent cellular immunity to the encoded antigen and more effectively prime strong antibody responses to an envelope protein boost, while circumventing anti-Ad immunity than a nonreplicating Ad recombinant. Therefore, we compared the priming ability and immune responses elicited by low-dose replicating and higher-dose nonreplicating Ad recombinants in chimpanzees, permissive for replication of human Ad, to allow evaluation of both vectors. To evaluate priming of antibody responses, we included boosts with oligomeric HIVSF162 gp140ΔV2, an HIV envelope subunit now being tested in phase I clinical trials. This optimally modified HIV envelope immunogen possesses a trimeric structure, has critical neutralizing epitopes exposed, and elicits antibodies able to neutralize a spectrum of clade B primary isolates (2, 37). In parallel, we assessed induction of humoral and cellular immune responses to the Ad vectors themselves.

HIV continues to be a major health problem world wide, however the situation is particularly serious in Asian and Sub-Saharan countries. Therefore, development of an effective HIV vaccine could help to reduce the severity of the disease and prevent infection. Over the last two decades significant efforts have been made towards inducing potent humoral and cellular immune responses by vaccination, however antibodies and CTL responses alone are likely not sufficient for inducing sterilizing immunity or long-term control of viral replication. Therefore, it is generally believed that both humoral and cellular responses will be needed for an effective HIV vaccine. In support of humoral immunity, monoclonal antibodies that recognize critical neutralizing epitopes have shown to be effective at passive transfer experiments in conferring protection against challenge infection. However, antibodies to similar epitope specificities are difficult to induce by vaccination. Therefore, optimization of Env structure is needed for exposing appropriate neutralizing epitopes and masking non-neutralizing epitopes. Since the crystal structure of the core of Env glycoprotein has been solved, efforts are in progress to design novel Env immunogens that may induce effective neutralizing responses. Furthermore, there are HIV-1 strains that are resistant to neutralization by monoclonal antibodies, yet neutralized by pooled sera from HIV-1 patients. Therefore, efforts should be made to identify these novel epitopes and to design strategies to incorporate them in potential vaccines. To facilitate comparative evaluation of vaccine immunogens for their ability to induce cross clade neutralizing antibodies, efforts should be made to use standardized neutralization assays and standard virus panels. Once potent HIV Env structure have been identified, their effectiveness may be enhanced through the use of adjuvants, delivery systems and prime and boost strategies to improve the quality and magnitude of neutralizing responses.

Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self-administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immunopotentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co-administered with pathogen-derived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administration. Although vaccines can be quite diverse in their composition, including inactivated virus, virus-like particles and inactivated bacteria (which are inert), protein-like vaccines, and non-replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus-based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines.

To evaluate the delivery of a novel HIV-1 antigen (gp120dV2 SF162) by surface adsorption or encapsulation within polylactide-co-glycolide microparticles and to compare both the formulations for their ability to preserve functional activity as measured by binding to soluble CD4.Poly(lactide-co-glycolide) microparticles were synthesized by a water-in-oil-in-water (w/o/w) emulsification method in the presence of the anionic surfactant dioctylsulfosuccinate (DSS) or polyvinyl alcohol. The HIV envelope glyocoprotein was adsorbed and encapsulated in the PLG particles. Binding efficiency and burst release measured to determine adsorption characteristics. The ability to bind CD4 was assayed to measure the functional integrity of gp120dV2 following different formulation processes.Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as hydrophobic attraction and structural accommodation of the polymer and biomolecule. The functional activity as measured by the ability of gp120dV2 to bind CD4 was maintained by adsorption onto anionic microparticles but drastically reduced by encapsulation.The antigen on the adsorbed PLG formulation maintained its binding ability to soluble CD4 in comparison to encapsulation, demonstrating the feasibility of using these novel anionic microparticles as a potential vaccine delivery system.

To understand the mechanisms involved in maintaining long-term immunological memory following mucosal immunizations, we determined the quality of serum hapten-specific immunoglobulins (Ig) and localized Ig-secreting cells (SC) of various isotypes in acute, persistent/resting memory and effector memory phases following oral versus intra-muscular (IM) immunizations. In the acute phase, both oral and IM immunizations induced high avidity Ig. However, in the persistent/resting memory phase, oral immunizations induced low avidity Ig while IM immunizations induced high avidity Ig. Following oral immunizations, in the persistent/resting memory phase, hapten-specific IgM titers in serum and IgM-SC in bone marrow (BM) dominated the immune response, suggesting an important role for IgM in the maintenance of memory.

The potential of vaccine-elicited anti-HIV envelope antibodies to control HIV-infection was evaluated by immunizing macaques with the HIV envelope protein and transiently depleting them of their CD8+ cells before intravenous challenge with the pathogenic CCR5-tropic SIV/HIV chimeric virus, SHIVSF162P4. Although sterilizing immunity was not achieved, all vaccinated animals effectively controlled infection and remained free of disease for the duration of observation (over 3 years). In contrast, during the same period, the control animals progressed to disease. Both the vaccinees and the controls developed robust cell-mediated antiviral and neutralizing antibody responses following infection. A comparative analysis of these responses suggests that the more effective long-term control of infection by the vaccinated animals is due to the more rapid development of anti-HIV envelope antibodies. These studies suggest that priming by vaccination of B cell anti-HIV envelope responses maybe crucial for the long-term control of HIV infection.

A HIV Vaccine, particularly that based on recombinant proteins and plasmid DNA, is likely to be less reactogenic than traditional vaccines, but also less immunogenic. Therefore, there is an urgent need for the development of new and improved adjuvants and delivery system for combination with HIV vaccine antigens. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; "vaccine delivery systems" and "immunostimulatory adjuvants". Vaccine delivery systems are generally particulate formulations e.g. emulsions, microparticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. The discovery of more potent adjuvants may allow the development of prophylactic and therapeutic vaccines against HIV. In addition, new adjuvants may also allow vaccines to be delivered mucosally.

Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1US4. We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (Rh) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smallerRhof the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.

A putative novel rhabdovirus (SfRV) was previously identified in a Spodoptera frugiperda cell line (Sf9 cells [ATCC CRL-1711 lot 58078522]) by next generation sequencing and extensive bioinformatic analysis. We performed an extensive analysis of our Sf9 cell bank (ATCC CRL-1711 lot 5814 [Sf9L5814]) to determine whether this virus was already present in cells obtained from ATCC in 1987. Inverse PCR of DNA isolated from Sf9 L5814 cellular DNA revealed integration of SfRV sequences in the cellular genome. RT-PCR of total RNA showed a deletion of 320 nucleotides in the SfRV RNA that includes the transcriptional motifs for genes X and L. Concentrated cell culture supernatant was analyzed by sucrose density gradient centrifugation and revealed a single band at a density of 1.14 g/ml. This fraction was further analysed by electron microscopy and showed amorphous and particulate debris that did not resemble a rhabdovirus in morphology or size. SDS-PAGE analysis confirmed that the protein composition did not contain the typical five rhabdovirus structural proteins and LC-MS/MS analysis revealed primarily of exosomal marker proteins, the SfRV N protein, and truncated forms of SfRV N, P, and G proteins. The SfRV L gene fragment RNA sequence was recovered from the supernatant after ultracentrifugation of the 1.14 g/ml fraction treated with diethyl ether suggesting that the SfRV L gene fragment sequence is not associated with a diethyl ether resistant nucleocapsid. Interestingly, the 1.14 g/ml fraction was able to transfer baculovirus DNA into Sf9L5814 cells, consistent with the presence of functional exosomes. Our results demonstrate the absence of viral particles in ATCC CRL-1711 lot 5814 Sf9 cells in contrast to a previous study that suggested the presence of infectious rhabdoviral particles in Sf9 cells from a different lot. This study highlights how cell lines with different lineages may present different virosomes and therefore no general conclusions can be drawn across Sf9 cells from different laboratories.

Titer on Chip (Flu-ToC) is a new technique for quantification of influenza hemagglutinin (HA) concentration. In order to evaluate the potential of this new technique, a comparison of Flu-ToC to more conventional methods was conducted using recombinant HA produced in a baculovirus expression system as a test case. Samples from current vaccine strains were collected from four different steps in the manufacturing process. A total of 19 samples were analysed by Flu-ToC (blinded), single radial immunodiffusion (SRID), an enzyme-linked immunosorbent assay (ELISA), and the purity adjusted bicinchoninic acid assay (paBCA). The results indicated reasonable linear correlation between Flu-ToC and SRID, ELISA, and paBCA, with regression slopes of log-log plots being 0.91, 1.03, and 0.91, respectively. The average ratio for HA content measured by Flu-ToC relative to SRID, ELISA, and paBCA was 83%, 147%, and 81%, respectively; indicating nearly equivalent potency determination for Flu-ToC relative to SRID and paBCA. These results, combined with demonstrated multiplexed analysis of all components within a quadrivalent formulation and robust response to HA strains over a wide time period, support the conclusion that Flu-ToC can be used as a reliable and time-saving alternative potency assay for influenza vaccines.

The recombinant hemagglutinin (rHA)-based influenza vaccine Flublok® has recently been approved in the United States as an alternative to the traditional egg-derived flu vaccines. Flublok is a purified vaccine with a hemagglutinin content that is threefold higher than standard inactivated influenza vaccines. When rHA derived from an H3N2 influenza virus was expressed, purified, and stored for 1 month, a rapid loss of in vitro potency (~50%) was observed as measured by the single radial immunodiffusion (SRID) assay. A comprehensive characterization of the rHA protein antigen was pursued to identify the potential causes and mechanisms of this potency loss. In addition, the biophysical and chemical stability of the rHA in different formulations and storage conditions was evaluated over time. Results demonstrate that the potency loss over time did not correlate with trends in changes to the higher order structure or hydrodynamic size of the rHA. The most likely mechanism for the early loss of potency was disulfide-mediated cross-linking of rHA, as the formation of non-native disulfide-linked multimers over time correlated well with the observed potency loss. Furthermore, a loss of free thiol content, particularly in specific cysteine residues in the antigen's C-terminus, was correlated with potency loss measured by SRID.

A combination of atovaquone and proguanil has been found to be quite effective in treating malaria, with little evidence of the emergence of resistance when atovaquone was used as a single agent. We have examined possible mechanisms for the synergy between these two drugs. While proguanil by itself had no effect on electron transport or mitochondrial membrane potential (ΔΨ m ), it significantly enhanced the ability of atovaquone to collapse ΔΨ m when used in combination. This enhancement was observed at pharmacologically achievable doses. Proguanil acted as a biguanide rather than as its metabolite cycloguanil (a parasite dihydrofolate reductase [DHFR] inhibitor) to enhance the atovaquone effect; another DHFR inhibitor, pyrimethamine, also had no enhancing effect. Proguanil-mediated enhancement was specific for atovaquone, since the effects of other mitochondrial electron transport inhibitors, such as myxothiazole and antimycin, were not altered by inclusion of proguanil. Surprisingly, proguanil did not enhance the ability of atovaquone to inhibit mitochondrial electron transport in malaria parasites. These results suggest that proguanil in its prodrug form acts in synergy with atovaquone by lowering the effective concentration at which atovaquone collapses ΔΨ m in malaria parasites. This could explain the paradoxical success of the atovaquone-proguanil combination even in regions where proguanil alone is ineffective due to resistance. The results also suggest that the atovaquone-proguanil combination may act as a site-specific uncoupler of parasite mitochondria in a selective manner.

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.

Methods Here, we designed disulfide-stabilized recombinant HIV1 subtype B (SF162) envelope glycoproteins (Env), gp120 and gp140, by insertion of site-specific cysteine pairs between two layers (layer 1 and 2) in inner domain of gp120. In addition, we identified a novel adjuvant approach using Carbopol 971P, a cross-linked polyanionic carbomer, in combination with the Novartis proprietary oil-in water adjuvant, MF59, to augment humoral immune responses to the Env glycoprotein. We performed thorough in vitro analysis of the disulfide-stabilized Env glycoprotein followed by in vivo evaluations of the adjuvanted-Env glycoprotein boost in rabbits.