Large-scale antibody immune response mapping of splenic B cells and bone marrow plasma cells in a transgenic mouse model.

Molecular characterization of antibody immunity and human antibody discovery is mainly carried out using peripheral memory B cells, and occasionally plasmablasts, that express B cell receptors (BCRs) on their cell surface. Despite the importance of plasma cells (PCs) as the dominant source of circulating antibodies in serum, PCs are rarely utilized because they do not express surface BCRs and cannot be analyzed using antigen-based fluorescence-activated cell sorting. Here, we studied the antibodies encoded by the entire mature B cell populations, including PCs, and compared the antibody repertoires of bone marrow and spleen compartments elicited by immunization in a human immunoglobulin transgenic mouse strain. To circumvent prior technical limitations for analysis of plasma cells, we applied single-cell antibody heavy and light chain gene capture from the entire mature B cell repertoires followed by yeast display functional analysis using a cytokine as a model immunogen. We performed affinity-based sorting of antibody yeast display libraries and large-scale next-generation sequencing analyses to follow antibody lineage performance, with experimental validation of 76 monoclonal antibodies against the cytokine antigen that identified three antibodies with exquisite double-digit picomolar binding affinity. We observed that spleen B cell populations generated higher affinity antibodies compared to bone marrow PCs and that antigen-specific splenic B cells had higher average levels of somatic hypermutation. A degree of clonal overlap was also observed between bone marrow and spleen antibody repertoires, indicating common origins of certain clones across lymphoid compartments. These data demonstrate a new capacity to functionally analyze antigen-specific B cell populations of different lymphoid organs, including PCs, for high-affinity antibody discovery and detailed fundamental studies of antibody immunity.

Characterization of prefusion-F-specific antibodies elicited by natural infection with human metapneumovirus.

Human metapneumovirus (hMPV) is a major cause of acute respiratory infections in infants and older adults, for which no vaccines or therapeutics are available. The viral fusion (F) glycoprotein is required for entry and is the primary target of neutralizing antibodies; however, little is known about the humoral immune response generated from natural infection. Here, using prefusion-stabilized F proteins to interrogate memory B cells from two older adults, we obtain over 700 paired non-IgM antibody sequences representing 563 clonotypes, indicative of a highly polyclonal response. Characterization of 136 monoclonal antibodies reveals broad recognition of the protein surface, with potently neutralizing antibodies targeting each antigenic site. Cryo-EM studies further reveal two non-canonical sites and the molecular basis for recognition of the apex of hMPV F by two prefusion-specific neutralizing antibodies. Collectively, these results provide insight into the humoral response to hMPV infection in older adults and will help guide vaccine development.

Pan-SARS neutralizing responses after third boost vaccination in non-human primate immunogenicity model.

The emergence of SARS-CoV-2 variants, especially Beta and Delta, has raised concerns about the reduced protection from previous infection or vaccination based on the original Wuhan-Hu-1 (D614) virus. To identify promising regimens for inducing neutralizing titers towards new variants, we evaluated monovalent and bivalent mRNA vaccines either as primary vaccination or as a booster in nonhuman primates (NHPs). Two mRNA vaccines, D614-based MRT5500 and Beta-based MRT5500ß, tested in sequential regimens or as a bivalent combination in naïve NHPs produced modest neutralizing titers to heterologous variants. However, when mRNA vaccines were administered as a booster to pre-immune NHPs, we observed a robust increase in neutralizing titers with expanded breadth towards all tested variants, and notably SARS-CoV-1. The breadth of the neutralizing response was independent of vaccine sequence or modality, as we further showed either MRT5500 or recombinant subunit Spike protein (with adjuvant) can serve as boosters to induce broadly neutralizing antibodies in the NHPs primed with MRT5500. The data support the notion that a third vaccination is key to boosting existing titers and improving the breadth of antibodies to address variants of concern, including those with an E484K mutation in the Receptor Binding Domain (RBD) (Beta, Gamma).

Development of multivalent mRNA vaccine candidates for seasonal or pandemic influenza.

Recent approval of mRNA vaccines for emergency use against COVID-19 is likely to promote rapid development of mRNA-based vaccines targeting a wide range of infectious diseases. Compared to conventional approaches, this vaccine modality promises comparable potency while substantially accelerating the pace of development and deployment of vaccine doses. Already demonstrated successfully for single antigen vaccines such as for COVID-19, this technology could be optimized for complex multi-antigen vaccines. Herein, utilizing multiple influenza antigens, we demonstrated the suitability of the mRNA therapeutic (MRT) platform for such applications. Seasonal influenza vaccines have three or four hemagglutinin (HA) antigens of different viral subtypes. In addition, influenza neuraminidase (NA), a tetrameric membrane protein, is identified as an antigen that has been linked to protective immunity against severe viral disease. We detail the efforts in optimizing formulations of influenza candidates that use unmodified mRNA encoding full-length HA or full-length NA encapsulated in lipid nanoparticles (LNPs). HA and NA mRNA-LNP formulations, either as monovalent or as multivalent vaccines, induced strong functional antibody and cellular responses in non-human primates and such antigen-specific antibody responses were associated with protective efficacy against viral challenge in mice.

Influenza vaccination in the elderly boosts antibodies against conserved viral proteins and egg-produced glycans.

Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galß1-4GalNAcß), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.

Optimization of the Codon Pair Usage of Human Respiratory Syncytial Virus Paradoxically Resulted in Reduced Viral Replication In Vivo and Reduced Immunogenicity.

We subjected various open reading frames (ORFs) in the genome of respiratory syncytial virus (RSV) to codon pair optimization (CPO) by increasing the content of codon pairs that are overrepresented in the human genome without changing overall codon usage and amino acid sequences. CPO has the potential to increase the expression of the encoded protein(s). Four viruses were made: Max A (with CPO of NS1, NS2, N, P, M, and SH ORFs), Max B (with CPO of G and F), Max L (with CPO of L), and Max FLC (with CPO of all ORFs except M2-1 and M2-2). Because of the possibility of increased viral replication, each CPO virus was attenuated by the inclusion of a codon deletion mutation (Δ1313) and a missense mutation (I1314L) in the L polymerase. CPO had no effect on multicycle virus replication in vitro, temperature sensitivity, or specific infectivity. Max A and L, which in common had CPO of one or more ORFs of proteins of the polymerase complex, exhibited global increases in viral protein synthesis. Max B alone exhibited decreased protein synthesis, and it alone had reduced single-cycle virus replication in vitro All CPO RSVs exhibited marginal reductions in replication in mice and hamsters. Surprisingly, the CPO RSVs induced lower levels of serum RSV-neutralizing antibodies in hamsters. This reduced immunogenicity might reflect reduced viral replication and possibly also the decrease in CpG and UpA dinucleotides as immune stimulators. Overall, our study describes paradoxical effects of CPO of an RNA virus on viral replication and the adaptive humoral immune response.IMPORTANCE Using computer algorithms and large-scale DNA synthesis, one or more ORFs of a microbial pathogen can be recoded by different strategies that involve the introduction of up to thousands of nucleotide changes without affecting amino acid coding. This approach has been used mostly to generate deoptimized viruses used as vaccine candidates. However, the effects of the converse approach of generating optimized viruses are still largely unknown. Here, various ORFs in the genome of respiratory syncytial virus (RSV) were codon pair optimized (CPO) by increasing the content of codon pairs that are overrepresented in the human genome. CPO did not affect RSV replication in multicycle replication experiments in vitro. However, replication was marginally reduced in two rodents models. In hamsters, CPO RSVs induced lower levels of serum RSV-neutralizing antibodies. Thus, CPO of an RNA virus for a mammalian host has paradoxical effects on virus replication and the adaptive humoral immune response.

Comparison of adjuvants to optimize influenza neutralizing antibody responses.

Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans.

A computationally designed H5 antigen shows immunological breadth of coverage and protects against drifting avian strains.

Since the first identification of the H5N1 Goose/Guangdong lineage in 1996, this highly pathogenic avian influenza virus has spread worldwide, becoming endemic in domestic poultry. Sporadic transmission to humans has raised concerns of a potential pandemic and underscores the need for a broad cross-protective influenza vaccine. Here, we tested our previously described methodology, termed Computationally Optimized Broadly Reactive Antigen (COBRA), to generate a novel hemagglutinin (HA) gene, termed COBRA-2, that was based on H5 HA sequences from 2005 to 2006. The COBRA-2 HA virus-like particle (VLP) vaccines were used to vaccinate chickens and the immune responses were compared to responses elicited by VLP's expressing HA from A/whooper swan/Mongolia/244/2005 (WS/05), a representative 2005 vaccine virus from clade 2.2. To support this evaluation a hemagglutination inhibition (HAI) breadth panel was developed consisting of phylogenetically and antigenically diverse H5 strains in circulation from 2005 to 2006, as well as recent drift variants (2008 - 2014). We found that the COBRA-2 VLP vaccines elicited robust HAI titers against this entire breadth panel, whereas the VLP vaccine based upon the recommended WS/05 HA only elicited HAI responses against a subset of strains. Furthermore, while all vaccines protected chickens against challenge with the WS/05 virus, only the human COBRA-2 VLP vaccinated birds were protected (80%) against a recent drifted clade 2.3.2.1B, A/duck/Vietnam/NCVD-672/2011 (VN/11) virus. This is the first report to demonstrate seroprotective antibody responses against genetically diverse clades and sub-clades of H5 viruses and protective efficacy against a recent drifted variant using a globular head based design strategy.

Elicitation of Protective Antibodies against 20 Years of Future H3N2 Cocirculating Influenza Virus Variants in Ferrets Preimmune to Historical H3N2 Influenza Viruses.

The vast majority of people already have preexisting immune responses to influenza viruses from one or more subtypes. However, almost all preclinical studies evaluate new influenza vaccine candidates in immunologically naive animals. Recently, our group demonstrated that priming naive ferrets with broadly reactive H1 COBRA HA-based vaccines boosted preexisting antibodies induced by wild-type H1N1 virus infections. These H1 COBRA hemagglutinin (HA) antigens induced antibodies with HAI activity against multiple antigenically different H1N1 viral variants. In this study, ferrets, preimmune to historical H3N2 viruses, were vaccinated with virus-like particle (VLP) vaccines expressing either an HA from a wild-type H3 influenza virus or a COBRA H3 HA antigen (T6, T7, T10, or T11). The elicited antisera had the ability to neutralize virus infection against either a panel of viruses representing vaccine strains selected by the World Health Organization or a set of viral variants that cocirculated during the same time period. Preimmune animals vaccinated with H3 COBRA T10 HA antigen elicited sera with higher hemagglutination inhibition (HAI) antibody titers than antisera elicited by VLP vaccines with wild-type HA VLPs in preimmune ferrets. However, while the T11 COBRA vaccine did not elicit HAI activity, the elicited antibodies did neutralize antigenically distinct H3N2 influenza viruses. Overall, H3 COBRA-based HA vaccines were able to neutralize both historical H3 and contemporary, as well as future, H3N2 viruses with higher titers than vaccines with wild-type H3 HA antigens. This is the first report demonstrating the effectiveness of a broadly reactive H3N3 vaccine in a preimmune ferret model.IMPORTANCE After exposure to influenza virus, the host generates neutralizing anti-hemagglutinin (anti-HA) antibodies against that specific infecting influenza strain. These antibodies can also neutralize some, but not all, cocirculating strains. The goal of next-generation influenza vaccines, such as HA head-based COBRA, is to stimulate broadly protective neutralizing antibodies against all strains circulating within a subtype, in particular those that persist over multiple influenza seasons, without requiring an update to the vaccine. To mimic the human condition, COBRA HA virus-like particle vaccines were tested in ferrets that were previously exposed to historical H3N2 influenza viruses. In this model, these vaccines elicited broadly protective antibodies that neutralized cocirculating H3N2 influenza viruses isolated over a 20-year period. This is the first study to show the effectiveness of H3N3 COBRA HA vaccines in a host with preexisting immunity to influenza.

Development of a Pan-H1 Influenza Vaccine.

The efficacy of current seasonal influenza vaccines varies greatly, depending on the match to circulating viruses. Although most vaccines elicit strain-specific responses, some present cross-reactive epitopes that elicit antibodies against diverse viruses and remain unchanged and effective for several years. To determine whether combinations of specific H1 hemagglutinin (HA) antigens stimulate immune responses that protect against diverse H1 influenza viruses, we evaluated the antibody responses elicited by HA-ferritin nanoparticles derived from six evolutionarily divergent H1 sequences and two computationally optimized broadly reactive antigen (COBRA) HA antigens. Humoral responses were assessed against a panel of 16 representative influenza virus strains from the past 80 years. HAs from the strains A/NewCaledonia/20/1999 (NC99), A/California/04/2009 (CA09), A/HongKong/117/1977 (HK77), COBRA X6, or P1 elicited neutralization against diverse strains, and a combination of three wild-type HA or two COBRA HA nanoparticles conferred significant additional breadth beyond that observed with any individual strain. Therefore, combinations of H1 HAs may constitute a pan-H1 influenza vaccine.IMPORTANCE Seasonal influenza vaccines elicit strain-specific immune responses designed to protect against circulating viruses. Because these vaccines often show limited efficacy, the search for a broadly protective seasonal vaccine remains a priority. Among different influenza virus subtypes, H1N1 has long been circulating in humans and has caused pandemic outbreaks. In order to assess the potential of a multivalent HA combination vaccine to improve the breadth of protection against divergent H1N1 viruses, HA-ferritin nanoparticles were made and evaluated in mice against a panel of historical and contemporary influenza virus strains. Trivalent combinations of H1 nanoparticles improved the breadth of immunity against divergent H1 influenza viruses.

Effect of genetic background and delivery route on the preclinical properties of a live attenuated RSV vaccine.

A safe and effective vaccine against RSV remains an important unmet public health need. Intranasally (IN) delivered live-attenuated vaccines represent the most extensively studied approach for immunization of RSV-naïve infants and children, however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we report pre-clinical immunogenicity and efficacy data utilizing a live-attenuated vaccine candidate, RGΔM2-2, which was obtained by deleting the M2-2 open reading frame from the genome of the MSA1 clinical isolate. Intramuscular (IM) administration of RGΔM2-2 in cotton rats induced immunity and protective efficacy that was comparable to that induced by intranasal (IN) immunization. In contrast, the protective efficacy of RGΔM2-2 delivered by the IM route to African green monkeys was substantially reduced as compared to the efficacy following IN administration, despite comparable levels of serum neutralizing antibodies. This result suggests that mucosal immunity may play an important role in RSV protection. The RGΔM2-2 vaccine also demonstrated different attenuation profiles when tested in cotton rats, non-human primates, and a human airway epithelial (HAE) cell model. The data suggest RGΔM2-2 is less attenuated than a similarly designed vaccine candidate constructed on the A2 genetic background. These findings have important implications with regard to both the design and the preclinical safety testing of live-attenuated vaccines.

Elicitation of Protective Antibodies against a Broad Panel of H1N1 Viruses in Ferrets Preimmune to Historical H1N1 Influenza Viruses.

Most preclinical animal studies test influenza vaccines in immunologically naive animal models, even though the results of vaccination may not accurately reflect the effectiveness of vaccine candidates in humans that have preexisting immunity to influenza. In this study, novel, broadly reactive influenza vaccine candidates were assessed in preimmune ferrets. These animals were infected with different H1N1 isolates before being vaccinated or infected with another influenza virus. Previously, our group has described the design and characterization of computationally optimized broadly reactive hemagglutinin (HA) antigens (COBRA) for H1N1 isolates. Vaccinating ferrets with virus-like particle (VLP) vaccines expressing COBRA HA proteins elicited antibodies with hemagglutination inhibition (HAI) activity against more H1N1 viruses in the panel than VLP vaccines expressing wild-type HA proteins. Specifically, ferrets infected with the 1986 virus and vaccinated with a single dose of the COBRA HA VLP vaccines elicited antibodies with HAI activity against 11 to 14 of the 15 H1N1 viruses isolated between 1934 and 2013. A subset of ferrets was infected with influenza viruses expressing the COBRA HA antigens. These COBRA preimmune ferrets had superior breadth of HAI activity after vaccination with COBRA HA VLP vaccines than COBRA preimmune ferrets vaccinated with VLP vaccines expressing wild-type HA proteins. Overall, priming naive ferrets with COBRA HA based viruses or using COBRA HA based vaccines to boost preexisting antibodies induced by wild-type H1N1 viruses, COBRA HA antigens elicited sera with the broadest HAI reactivity against multiple antigenic H1N1 viral variants. This is the first report demonstrating the effectiveness of a broadly reactive or universal influenza vaccine in a preimmune ferret model.IMPORTANCE Currently, many groups are testing influenza vaccine candidates to meet the challenge of developing a vaccine that elicits broadly reactive and long-lasting protective immune responses. The goal of these vaccines is to stimulate immune responses that react against most, if not all, circulating influenza strains, over a long period of time in all populations of people. Commonly, these experimental vaccines are tested in naive animal models that do not have anti-influenza immune responses; however, humans have preexisting immunity to influenza viral antigens, particularly antibodies to the HA and NA glycoproteins. Therefore, this study investigated how preexisting antibodies to historical influenza viruses influenced HAI-specific antibodies and protective efficacy using a broadly protective vaccine candidate.

Computationally Optimized Broadly Reactive Hemagglutinin Elicits Hemagglutination Inhibition Antibodies against a Panel of H3N2 Influenza Virus Cocirculating Variants.

Each influenza season, a set of wild-type viruses, representing one H1N1, one H3N2, and one to two influenza B isolates, are selected for inclusion in the annual seasonal influenza vaccine. In order to develop broadly reactive subtype-specific influenza vaccines, a methodology called computationally optimized broadly reactive antigens (COBRA) was used to design novel hemagglutinin (HA) vaccine immunogens. COBRA technology was effectively used to design HA immunogens that elicited antibodies that neutralized H5N1 and H1N1 isolates. In this report, the development and characterization of 17 prototype H3N2 COBRA HA proteins were screened in mice and ferrets for the elicitation of antibodies with HA inhibition (HAI) activity against human seasonal H3N2 viruses that were isolated over the last 48 years. The most effective COBRA HA vaccine regimens elicited antibodies with broader HAI activity against a panel of H3N2 viruses than wild-type H3 HA vaccines. The top leading COBRA HA candidates were tested against cocirculating variants. These variants were not efficiently detected by antibodies elicited by the wild-type HA from viruses selected as the vaccine candidates. The T-11 COBRA HA vaccine elicited antibodies with HAI and neutralization activity against all cocirculating variants from 2004 to 2007. This is the first report demonstrating broader breadth of vaccine-induced antibodies against cocirculating H3N2 strains compared to the wild-type HA antigens that were represented in commercial influenza vaccines.IMPORTANCE There is a need for an improved influenza vaccine that elicits immune responses that recognize a broader number of influenza virus strains to prevent infection and transmission. Using the COBRA approach, a set of vaccines against influenza viruses in the H3N2 subtype was tested for the ability to elicit antibodies that neutralize virus infection against not only historical vaccine strains of H3N2 but also a set of cocirculating variants that circulated between 2004 and 2007. Three of the H3N2 COBRA vaccines recognized all of the cocirculating strains during this era, but the chosen wild-type vaccine strains were not able to elicit antibodies with HAI activity against these cocirculating strains. Therefore, the COBRA vaccines have the ability to elicit protective antibodies against not only the dominant vaccine strains but also minor circulating strains that can evolve into the dominant vaccine strains in the future.

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure.

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.

Core bead chromatography for preparation of highly pure, infectious respiratory syncytial virus in the negative purification mode.

Respiratory syncytial virus (RSV) is an important human pathogen, and is the most frequent viral cause of severe respiratory disease in infants. In addition, it is increasingly being recognized as an important cause of respiratory disease in the elderly and immunocompromised. Although a passive prophylactic treatment does exist for high-risk neonates and children, the overall disease burden warrants the development of a safe and effective prophylactic vaccine for use in otherwise healthy newborns and children. RSV is known to be an extremely labile virus, prone to aggregation and loss of infectious titer during virus handling and preparation procedures. To date infective RSV virions have been prepared by methods which are not readily scalable, such as density gradient ultracentrifugation. In this study we describe a scalable, chromatography-based purification procedure for preparation of highly pure, infectious RSV. The purification scheme is based on core bead technology and hollow fiber tangential flow filtration (TFF) and results in a ∼60% recovery of infectious virus titer. This method can be used to prepare highly purified wild type or live-attenuated vaccine strain viruses with titers as high as 1×10(8) plaque forming units per mL. A live-attenuated RSV vaccine prepared by this method was found to be immunogenic and protective in vivo, and its purity was 50-200-fold greater with respect to host cell dsDNA and Vero host cell proteins, than the raw feed stream. The results presented here can be considered a starting point for downstream process development of a live-attenuated vaccine approach for prevention of disease by RSV.

CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner.

Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein's CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma.

Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization.

Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32 °C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negative-strand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.

Immunogenicity and efficacy of intramuscular replication-defective and subunit vaccines against herpes simplex virus type 2 in the mouse genital model.

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted virus that is highly prevalent worldwide, causing a range of symptoms that result in significant healthcare costs and human suffering. ACAM529 is a replication-defective vaccine candidate prepared by growing the previously described dl5-29 on a cell line appropriate for GMP manufacturing. This vaccine, when administered subcutaneously, was previously shown to protect mice from a lethal vaginal HSV-2 challenge and to afford better protection than adjuvanted glycoprotein D (gD) in guinea pigs. Here we show that ACAM529 given via the intramuscular route affords significantly greater immunogenicity and protection in comparison with subcutaneous administration in the mouse vaginal HSV-2 challenge model. Further, we describe a side-by-side comparison of intramuscular ACAM529 with a gD vaccine across a range of challenge virus doses. While differences in protection against death are not significant, ACAM529 protects significantly better against mucosal infection, reducing peak challenge virus shedding at the highest challenge dose by over 500-fold versus 5-fold for gD. Over 27% (11/40) of ACAM529-immunized animals were protected from viral shedding while 2.5% (1/40) were protected by the gD vaccine. Similarly, 35% (7/20) of mice vaccinated with ACAM529 were protected from infection of their dorsal root ganglia while none of the gD-vaccinated mice were protected. These results indicate that measuring infection of the vaginal mucosa and of dorsal root ganglia over a range of challenge doses is more sensitive than evaluating survival at a single challenge dose as a means of directly comparing vaccine efficacy in the mouse vaginal challenge model. The data also support further investigation of ACAM529 for prophylaxis in human subjects.