The RSV F p27 peptide: current knowledge, important questions.

Respiratory syncytial virus (RSV) remains a leading cause of hospitalizations and death for young children and adults over 65. The worldwide impact of RSV has prioritized the search for an RSV vaccine, with most targeting the critical fusion (F) protein. However, questions remain about the mechanism of RSV entry and RSV F triggering and fusion promotion. This review highlights these questions, specifically those surrounding a cleaved 27 amino acids long peptide within F, p27.

The Efficiency of p27 Cleavage during In Vitro Respiratory Syncytial Virus (RSV) Infection Is Cell Line and RSV Subtype Dependent.

Respiratory syncytial virus (RSV) fusion protein (F) is highly conserved between subtypes A and B (RSV/A and RSV/B). To become fully active, F precursor undergoes enzymatic cleavage to yield F1 and F2 subunits and releases a 27-amino-acid peptide (p27). Virus-cell fusion occurs when RSV F undergoes a conformational change from pre-F to post-F. Previous data show that p27 is detected on RSV F, but questions remain regarding if and how p27 affects the conformation of mature RSV F. Monoclonal antibodies against p27, site Ø (pre-F specific), and site II were used to monitor RSV F conformation by enzyme-linked immunosorbent assay (ELISA) and imaging flow cytometry. Pre-F to post-F conformational change was induced by a temperature stress test. We found that p27 cleavage efficiency was lower on sucrose-purified RSV/A (spRSV/A) than on spRSV/B. In addition, cleavage of RSV F was cell line dependent: HEp-2 cells had higher retention of p27 than did A549 cells when infected with RSV. Higher levels of p27 were also found on RSV/A-infected cells than on RSV/B-infected cells. We observed that RSV/A F with higher p27 levels could better sustain the pre-F conformation during the temperature stress challenge in both spRSV- and RSV-infected cell lines. Our findings suggest that despite F sequence similarity, p27 of RSV subtypes was cleaved with different efficiencies, which were also dependent on the cell lines used for infection. Importantly, the presence of p27 was associated with greater stability of the pre-F conformation, supporting the possibility that RSV has more than one mechanism for fusion to the host cell. IMPORTANCE RSV fusion protein (F) plays an important role in entry and viral fusion to the host cell. The F undergoes proteolytic cleavages releasing a 27-amino-acid peptide (p27) to become fully functional. The role of p27 in viral entry and the function of the partially cleaved F containing p27 has been overlooked. p27 is thought to destabilize the F trimers, and thus, there is need for a fully cleaved F. In this study, we detected p27 on purified RSV virions and on the surface of virus-infected HEp-2 and A549 cells for circulating RSV strains of both subtypes. Higher levels of partially cleaved F containing p27 better sustained the pre-F conformation during the temperature stress challenge. Our findings highlight that the cleavage efficiency of p27 is different between RSV subtypes and among cell lines and that the presence of p27 contributes to the stability of the pre-F conformation.

Serum IgG anti-SARS-CoV-2 Binding Antibody Level Is Strongly Associated With IgA and Functional Antibody Levels in Adults Infected With SARS-CoV-2.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019 in Wuhan, China, and then rapidly spread causing an unprecedented pandemic. A robust serological assay is needed to evaluate vaccine candidates and better understand the epidemiology of coronavirus disease (COVID-19). Methods: We used the full-length spike (S) protein of SARS-CoV-2 for the development of qualitative and quantitative IgG and IgA anti-S enzyme linked immunosorbent assays (ELISA). A total of 320 sera used for assay development were comprised of pandemic sera from SARS-CoV-2 infected adults (n=51) and pre-pandemic sera (n=269) including sera from endemic human coronavirus infected adults. Reverse cumulative curves and diagnostic test statistics were evaluated to define the optimal serum dilution and OD cutoff value for IgG anti-S and IgA anti-S ELISAs. The IgG and IgA anti-S, and three functional antibodies (ACE-2 receptor blocking antibody, lentipseudovirus-S neutralizing antibody, and SARS-CoV-2 neutralizing antibody) were measured using additional SARS-CoV-2 PCR positive sera (n=76) and surveillance sera (n=25). Lastly, the IgG and IgA anti-S levels were compared in different demographic groups. Results: The optimal serum dilution for the qualitative IgG anti-S ELISA was at 1:1024 yielding a 99.6% specificity, 92.2% sensitivity, 92.9% positive predictive value (PPV), and 99.6% negative predictive value (NPV) at a SARS-CoV-2 seroprevalence of 5%. The optimal serum dilution for the qualitative IgA anti-S ELISA was at 1:128 yielding a 98.9% specificity, 76.5% sensitivity, 78.3% PPV, and 98.8% NPV at the same seroprevalence. Significant correlations were demonstrated between the IgG and IgA (r=0.833 for concentrations, r=0.840 for titers) as well as between IgG and three functional antibodies (r=0.811-0.924 for concentrations, r=0.795-0.917 for titers). The IgG and IgA anti-S levels were significantly higher in males than females (p<0.05), and in adults with moderate/severe symptoms than in adults with mild/moderate symptoms (p<0.001). Conclusion: We developed a highly specific and sensitive IgG anti-S ELISA assay to SARS-CoV-2 using full length S protein. The IgG anti-S antibody level was strongly associated with IgA and functional antibody levels in adults with SARS-CoV-2 infection. Gender and disease severity, rather than age, play an important role in antibody levels.

Profile of respiratory syncytial virus prefusogenic fusion protein nanoparticle vaccine.

INTRODUCTION: Respiratory Syncytial Virus (RSV) is a leading cause of acute lower respiratory infections worldwide. The RSV fusion (F) glycoprotein is a major focus of vaccine development. Despite over 60 years of research, there is no licensed vaccine for RSV. AREAS COVERED: The primary focus of this review is a novel RSV-F recombinant nanoparticle vaccine from Novavax utilizing the F protein, a conserved and immunodominant surface glycoprotein. This RSV F recombinant nanoparticle vaccine adsorbed to 0.4 mg of aluminum phosphate was ultimately administered by a single intramuscular injection during the third trimester of pregnancy in an effort to induce passive immunity in newborns. Its mechanism, performance in clinical trials, and place in RSV vaccine history are discussed. EXPERT OPINION: The vaccine was safe and well tolerated in pregnant women and the results suggest potential benefits with respect to other medically relevant end-point events involving RSV-associated respiratory and all-cause disease in infants. However, the RSV-F recombinant nanoparticle vaccine did not meet the pre-specified primary success criteria for efficacy against RSV-associated, medically significant lower respiratory tract infection in infants up to 90 days of life. The potential benefits to infants from maternal immunization and excellent safety profile warrant further confirmatory studies.

Antibody Response to the Furin Cleavable Twenty-Seven Amino Acid Peptide (p27) of the Fusion Protein in Respiratory Syncytial Virus (RSV) Infected Adult Hematopoietic Cell Transplant (HCT) Recipients.

Background: Cleavage of the inactive precursor fusion protein (F0) of respiratory syncytial virus (RSV) at two furin-recognition sites is required for membrane fusion activity, and the cleavage releases the twenty-seven amino acid peptide (p27). However, a recent study shows that p27 was an immunodominant epitope in RSV infected children, indicating that p27 was recognized as an immunogen. In the present study, we investigated the immunogenicity of p27 in an immunocompromised population of adults by measuring serum and mucosal antibody responses to p27 in samples from adult hematopoietic cell transplant (HCT) recipients. Methods: We prospectively enrolled a cohort of RSV infected HCT recipients. Serum and nasal-wash samples were obtained within the first week of RSV infection (acute) and 3 to 5 weeks post-infection (convalescent). We quantified the serum and mucosal IgG and IgA anti-p27 antibodies by a RSV/A p27 peptide enzyme-linked immunosorbent assay (ELISA) and serum and mucosal p27 like antibodies (P27LA) by a p27 competitive antibody (P27CA) assay. Results: The lower limit of detection for the ELISA and P27CA assays was 0.2 and 50 ng/mL, respectively with no cross-reaction detected with a panel of monoclonal antibodies targeting pre-fusion and post-fusion antigenic sites. P27 antibodies were detected at nanogram concentration in sera and nasal washes in the majority of RSV infected HCT recipients. However, there was no significant difference in the geometric mean antibody concentrations between the acute and convalescent sera (except for serum P27LA), between HCT recipients who shed RSV <14 days and ≥14 days, as well as between RSV/A and RSV/B infected HCT recipients. In addition, approximately 30% of HCT recipients had a 4-fold or greater decrease in mucosal IgG and IgA anti-p27 antibodies during viral clearance. Conclusion: In conclusion, in RSV naturally infected adult HCT recipients, the antibodies against p27 were detectable in both serum and nasal wash samples with higher concentration in serum than that in nasal washes. However, nearly 30% of RSV infected HCT recipients had a significant decrease in their mucosal anti-p27 antibody, suggesting that IgG and IgA anti-p27 antibodies were binding to either free viruses or RSV infected cells containing p27, and that anti-p27 antibodies in the respiratory tract were part of the mucosal antibody response in controlling RSV infection.

Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate.

From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast-Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.

Structure of SALO, a leishmaniasis vaccine candidate from the sand fly Lutzomyia longipalpis.

BACKGROUND: Immunity to the sand fly salivary protein SALO (Salivary Anticomplement of Lutzomyia longipalpis) protected hamsters against Leishmania infantum and L. braziliensis infection and, more recently, a vaccine combination of a genetically modified Leishmania with SALO conferred strong protection against L. donovani infection. Because of the importance of SALO as a potential component of a leishmaniasis vaccine, a plan to produce this recombinant protein for future scale manufacturing as well as knowledge of its structural characteristics are needed to move SALO forward for the clinical path. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant SALO was expressed as a soluble secreted protein using Pichia pastoris, rSALO(P), with yields of 1g/L and >99% purity as assessed by SEC-MALS and SDS-PAGE. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-Kalpha to 1.94 Å and refined to Rfactor 17%. SALO is ~80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. SALO has three predicted human CD4+ T cell epitopes on surface exposed helices. CONCLUSIONS/SIGNIFICANCE: The results indicate that SALO as expressed and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing. SALO has a novel structure, is not similar to any human proteins, is immunogenic in rodents, and does not have the anti-complement activity observed in the native salivary protein which are all important attributes to move this vaccine candidate forward to the clinical path.

A simple fluorescence-based assay for quantification of the Toll-Like Receptor agonist E6020 in vaccine formulations.

Despite the generally accepted immunostimulatory effect of Toll-Like Receptor 4 (TLR4) agonists and their value as vaccine adjuvants, there remains a demand for fast and easy quantification assays for these TLR4 agonists in order to accelerate and improve vaccine formulation studies. A new medium-throughput method was developed for the quantification of the TLR4 agonist, E6020, independent of the formulation composition. The assay uses a fluorescent hydrazide (DCCH) to label the synthetic lipopolysaccharide (LPS) analog E6020 through its diketone groups. This novel, low-cost, and fluorescence based assay may obviate the need for traditional approaches that primarily rely on Fourier transform infrared spectroscopy (FTIR) or mass spectrometry. The experiments were performed in a wide diversity of vaccine formulations containing E6020 to assess method robustness and accuracy. The assay was also expanded to evaluate the loading efficiency of E6020 in poly(lactic-co-glycolic acid) (PLGA) micro-particles.

Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

Optimization and revision of the production process of the Necator americanus glutathione S-transferase 1 (Na-GST-1), the lead hookworm vaccine recombinant protein candidate.

Infection by the human hookworm Necator americanus is a leading cause of anemia and disability in the developing countries of Africa, Asia, and the Americas. In order to prevent childhood hookworm disease in resource poor settings, a recombinant vaccine is under development by the Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, a Product Development Partnership (PDP). Previously, we reported on the expression and purification of a highly promising hookworm vaccine candidate, Na-GST-1, an N. americanus glutathione s-transferase expressed in Pichia pastoris (yeast), which led to production of 1.5 g of 95% pure recombinant protein at a 20L scale. (1) (,) (2) (,) (3) This yield and purity of Na-GST-1 was sufficient for early pilot manufacturing and initial phase 1 clinical testing. However, based on the number of doses which would be required to allow mass vaccination and a potential goal to deliver a vaccine as inexpensively as possible, a higher yield of expression of the recombinant antigen at the lowest possible cost is highly desirable. Here we report on modifications to the fermentation (upstream process) of the antigen expressed in P. pastoris, and to the purification (downstream process) of the recombinant protein that allowed for a 2-3-fold improvement in the final yield of Na-GST-1 purified protein. The major improvements included upstream process changes such as the addition of a sorbitol pulse and co-feed during methanol induction as well as an extension of the induction stage to approximately 96 hours; downstream process changes included modifying the UFDF to flat sheet with a 10 kDa Molecular Weight cut-off (MWCO), adjusting the capacity of an ion-exchange chromatography step utilizing a gradient elution as opposed to the original step elution, and altering the hydrophobic interaction chromatography conditions. The full process, as well as the purity and stability profiles of the target Na-GST-1, and its formulation on Alhydrogel(®), is described.

Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs.

Schistosomiasis is a parasitic disease that affects over 200 million people. Vaccine candidates have been identified, including Schistosoma mansoni venom allergen-like proteins (SmVALs) from the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. The first SmVAL structure, SmVAL4, was refined to a resolution limit of 2.16 Å. SmVAL4 has a unique structure that could not be predicted from homologous structures, with longer loops and an unusual C-terminal extension. SmVAL4 has the characteristic α/ß-sandwich and central SCP/TAPS cavity. Furthermore, SmVAL4 has only one of the signature CAP cavity tetrad amino-acid residues and is missing the histidines that coordinate divalent cations such as Zn(2+) in other SCP/TAPS proteins. SmVAL4 has a cavity between α-helices 1 and 4 that was observed to bind lipids in tablysin-15, suggesting the ability to bind lipids. Subsequently, SmVAL4 was shown to bind cholesterol in vitro. Additionally, SmVAL4 was shown to complement the in vivo sterol-export phenotype of yeast mutants lacking their endogenous CAP proteins. Expression of SmVAL4 in yeast cells lacking endogenous CAP function restores the block in sterol export. These studies suggest an evolutionarily conserved lipid-binding function shared by CAP proteins such as SmVAL4 and yeast CAP proteins such as Pry1.

Structural and biochemical analyses of alanine racemase from the multidrug-resistant Clostridium difficile strain 630.

Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. C. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. The severity of C. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. Since D-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistant C. difficile strain 630 are presented. Three crystal structures of C. difficile Alr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics for C. difficile infections.

Expression at a 20L scale and purification of the extracellular domain of the Schistosoma mansoni TSP-2 recombinant protein: a vaccine candidate for human intestinal schistosomiasis.

A novel recombinant protein vaccine for human schistosomiasis caused by Schistosoma mansoni is under development. The Sm-TSP-2 schistosomiasis vaccine is comprised of a 9 kDa recombinant protein corresponding to the extracellular domain of a unique S. mansoni tetraspanin. Here, we describe the cloning and the expression of the external loop of Sm-TSP-2 recombinant protein secreted by Pichia Pink the process development at 20L scale fermentation, and the two-steps purification, which resulted in a protein recovery yield of 31% and a protein purity of 97%. The developed processes are suitable for the production of purified protein for subsequent formulation and Phase 1 clinical studies.

Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis.

A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0-8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine.

Advances in vaccines against neglected tropical diseases: enhancing physical stability of a recombinant hookworm vaccine through biophysical and formulation studies.

A bivalent recombinant vaccine for human hookworm disease is under development. One of the lead candidate antigens in the vaccine is a glutathione S-transferase cloned from the hookworm Necator americanus (Na-GST-1) which is expressed in the yeast Pichia pastoris. Based on preliminary studies demonstrating that the recombinant protein was not stable in an acetate buffer at pH 6, we undertook an extensive stability analysis of the molecule. To improve and optimize stability we complemented traditional methods employed for macromolecule and vaccine stabilization with biophysical techniques that were incorporated into a systematic process based on an eigenvector approach. Large data sets, obtained from a variety of experimental methods were used to establish a color map ("empirical phase diagram") of the physical stability of the vaccine antigen over a wide range of temperature and pH. The resulting map defined "apparent phase boundaries" that were used to develop high throughput screening assays. These assays were then employed to identify excipients that stabilized the antigen against physical degradation that could otherwise result in losses of physicochemical integrity, immunogenicity, and potency of the vaccine. Based on these evaluations, the recombinant Na-GST-1 antigen was reformulated and ultimately produced under Good Manufacturing Practices and with an acceptable stability profile.