Mapping of a Novel H3-Specific Broadly Neutralizing Monoclonal Antibody Targeting the Hemagglutinin Globular Head Isolated from an Elite Influenza Virus-Immunized Donor Exhibiting Serological Breadth.

The discovery of potent and broadly protective influenza virus epitopes could lead to improved vaccines that are resistant to antigenic drift. Here, we describe human antibody C585, isolated from a vaccinee with remarkable serological breadth as measured by hemagglutinin inhibition (HAI). C585 binds and neutralizes multiple H3N2 strains isolated between 1968 and 2016, including strains that emerged up to 4 years after B cells were isolated from the vaccinated donor. The crystal structure of C585 Fab in complex with the HA from A/Switzerland/9715293/2013 (H3N2) shows that the antibody binds to a novel and well-conserved epitope on the globular head of H3 HA and that it differs from other antibodies not only in its epitope but in its binding geometry and hypermutated framework 3 region, thereby explaining its breadth and ability to mediate hemagglutination inhibition across decades of H3N2 strains. The existence of epitopes such as the one elucidated by C585 has implications for rational vaccine design.IMPORTANCE Influenza viruses escape immunity through continuous antigenic changes that occur predominantly on the viral hemagglutinin (HA). Induction of broadly neutralizing antibodies (bnAbs) targeting conserved epitopes following vaccination is a goal of universal influenza vaccines and advantageous in protecting hosts against virus evolution and antigenic drift. To date, most of the discovered bnAbs bind either to conserved sites in the stem region or to the sialic acid-binding pocket. Generally, antibodies targeting the stem region offer broader breadth with low potency, while antibodies targeting the sialic acid-binding pocket cover narrower breadth but usually have higher potency. In this study, we identified a novel neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against a broad range of H3N2 with high potency. This epitope may provide insights for future universal vaccine design.

Pre-existing Fc profiles shape the evolution of neutralizing antibody breadth following influenza vaccination.

Under the ever-present threat of a pandemic influenza strain, the evolution of a broadly reactive, neutralizing, functional, humoral immune response may hold the key to protection against both circulating and emerging influenza strains. We apply a systems approach to profile hemagglutinin- and neuraminidase-specific humoral signatures that track with the evolution of broad immunity in a cohort of vaccinated individuals and validate these findings in a second longitudinal cohort. Multivariate analysis reveals the presence of a unique pre-existing Fcγ-receptor-binding antibody profile in individuals that evolved broadly reactive hemagglutination inhibition activity (HAI), marked by the presence of elevated levels of pre-existing FCGR2B-binding antibodies. Moreover, vaccination with FCGR2B-binding antibody-opsonized influenza results in enhanced antibody titers and HAI activity in a murine model. Together, these data suggest that pre-existing FCGR2B binding antibodies are a key correlate of the evolution of broadly protective influenza-specific antibodies, providing insight for the design of next-generation influenza vaccines.

Novel structural insights for a pair of monoclonal antibodies recognizing non-overlapping epitopes of the glucosyltransferase domain of Clostridium difficile toxin B.

Clostridium difficile toxins are the primary causative agents for hospital-acquired diarrhea and pseudomembranous colitis. Numerous monoclonal antibodies (mAbs) targeting different domains of Clostridium difficile toxin have been reported. Here we report the crystal structures of two mAbs, B1 and B2, in complex with the glycosyltransferase domain (GTD) of the Clostridium difficile toxin B (TcdB). B2 bound to the N-terminal 4 helix bundle of the GTD, a conserved membrane localization domain (MLD) found in the large clostridial glycosylating toxin family implicated in targeting plasma membrane. B1 bound to a distinct epitope at the hinge region between the MLD and the catalytic subdomain of the GTD. Functional studies revealed the potency of these mAbs in vitro and in vivo to be synergistic when given in combination.

Immunogenicity and protective efficacy of RSV G central conserved domain vaccine with a prefusion nanoparticle.

Respiratory syncytial virus (RSV) G glycoprotein has recently reemerged as a vaccine antigen due to its ability to elicit potent neutralizing antibodies and ameliorate disease in animal models. Here we designed three constructs to display the G central conserved domain (Gcc) focused on inducing broad and potent neutralizing antibodies. One construct displaying Gcc from both RSV subgroups trimerized via a C-terminal foldon (Gcc-Foldon) was highly immunogenic in mice and in MIMIC, a pre-immune human in vitro model. To explore an optimal RSV vaccine, we combined the Gcc-Foldon antigen with a stabilized pre-fusion-F nanoparticle (pre-F-NP) as a bivalent vaccine and detected no antigenic interference between the two antigens in the MIMIC model. In RSV-primed macaques, the bivalent vaccine elicited potent humoral responses. Furthermore, both Gcc-Foldon and the bivalent vaccine conferred effective protection against RSV challenge in mice. This two-component vaccine could potentially provide effective protection against RSV infection in humans and warrants further clinical evaluation.

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.

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.

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.

Enzymatic mechanism of human apurinic/apyrimidinic endonuclease against a THF AP site model substrate.

The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo) is a major factor in the maintenance of the integrity of the human genome. There are estimates that this enzyme is responsible for eliminating as many as 10(5) potentially mutagenic and genotoxic lesions from the genome of each cell every day. Furthermore, inhibition of AP endonuclease may be effective in decreasing the dose requirements of chemotherapeutics used in the treatment of cancer as well as other diseases. Therefore, it is essential to accurately and directly characterize the enzymatic mechanism of AP endo. Here we describe specifically designed double-stranded DNA oligomers containing tetrahydrofuran (THF) with a 5'-phosphorothioate linkage as the abasic site substrate. Using H(2)(18)O during the cleavage reaction and leveraging the stereochemical preferences of AP endo and T4 DNA ligase for phosphorothioate substrates, we show that AP endo acts by a one-step associative phosphoryl transfer mechanism on a THF-containing substrate.

Deciphering the domain specificity of C. difficile toxin neutralizing antibodies.

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. The pathological effects of CDI are primarily attributed to toxins A (TcdA) and B (TcdB). Adequate toxin-specific antibody responses are associated with asymptomatic carriage, whereas insufficient humoral responses are associated with recurrent CDI. While the data supporting the importance of anti-toxin antibodies are substantial, clarity about the toxin domain specificity of these antibodies is more limited. To investigate this matter, combinations of human mAbs targeting multiple domains of TcdB were assessed using toxin neutralization assays. These data revealed that a combination of mAbs specific to all major toxin domains had improved neutralizing potency when compared to equivalent concentrations of a single mAb or a combination of mAbs against one or two domains. The function and toxin domain binding specificity of serum antibodies elicited by immunization of hamsters with a toxoid vaccine candidate was also assessed. Immunization with a toxoid vaccine candidate provoked toxin neutralizing antibodies specific to multiple domains of both TcdA and TcdB. When assessed in a toxin neutralization assay, polyclonal sera displayed greater activity against elevated concentrations of toxins than equivalent concentrations of individual mAbs. These data suggest a potential benefit of any antibody based therapeutic or prophylactic treatment that targets multiple toxin domains.

Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease.

AP endonuclease (AP endo), a key enzyme in repair of abasic sites in DNA, makes a single nick 5' to the phosphodeoxyribose of an abasic site (AP-site). We recently proposed a novel mechanism, whereby the enzyme uses a key tyrosine (Tyr(171)) to directly attack the scissile phosphate of the AP-site. We showed that loss of the tyrosyl hydroxyl from Tyr(171) resulted in dramatic diminution in enzymatic efficiency. Here we extend the previous work to compare binding/recognition of AP endo to oligomeric DNA with and without an AP-site by wild type enzyme and several tyrosine mutants including Tyr(128), Tyr(171) and Tyr(269). We used single turnover and electrophoretic mobility shift assays. As expected, binding to DNA with an AP-site is more efficient than binding to DNA without one. Unlike catalytic cleavage by AP endo, which requires both hydroxyl and aromatic moieties of Tyr(171), the ability to bind DNA efficiently without an AP-site is independent of an aromatic moiety at position 171. However, the ability to discriminate efficiently between DNA with and without an AP-site requires tyrosine at position 171. Thus, AP endo requires a tyrosine at the active site for the properties that enable it to behave as an efficient, processive endonuclease.

Impact of age and pre-existing influenza immune responses in humans receiving split inactivated influenza vaccine on the induction of the breadth of antibodies to influenza A strains.

Most humans have pre-existing immunity to influenza viruses. In this study, volunteers (ages of 18-85 years) were vaccinated with split, inactivated Fluzone™ influenza vaccine in four consecutive influenza seasons from 2013 to 2016 seasons. The impact of repeated vaccination on breadth and durability of antibodies was assessed as a result of vaccine strain changes. Total IgG anti-hemagglutinin (HA) binding antibodies and hemagglutination-inhibition (HAI) activity increased in all age groups against both influenza A HA components in the vaccine post-vaccination (day 21). However, younger subjects maintained seroprotective titers to the vaccine strains, which resulted in higher seroconversion rates in the elderly, since the HAI titers in elderly subjects were more likely to decline prior to the next season. Young subjects had significant HAI activity against historical, as well as contemporary H1 and H3 vaccine strains from the mid-1980s to present. In contrast, elderly subjects had HAI activity to H1 strains from all years, but were more likely to have HAI activity to older strains from 1918-1950s. They also had a more restricted HAI profile against H3 viruses compared to young subjects recognizing H3N2 influenza viruses from the mid-2000s to present. Vaccine recipients were then categorized by whether subjects seroconverted from a seronegative or seropositive pre-vaccination state. Regardless of age, immunological recall or 'back-boosting' to antigenically related strains were associated with seroconversion to the vaccine strain. Overall, both younger and older people have the ability to mount a breadth of immune responses following influenza vaccination. This report describes how imprinting exposure differs across age groups, influences antibody cross-reactivity to past hemagglutinin antigenic variants, and shapes immune responses elicited by current split inactivated influenza vaccines. Understanding how current influenza vaccines are influenced by pre-existing immunity in people of different ages is critical for designing the next-generation of 'universal' or broadly-protective influenza vaccines.

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.

Novel role of tyrosine in catalysis by human AP endonuclease 1.

Apurinic/apyrimidinic endonuclease (AP endo, HAP1) recognizes abasic sites in ds DNA and makes a single nick in the backbone 5' to the abasic site. In this report we examine the roles of three conserved tyrosine residues in close proximity to the active site. We show that Tyr(128) and Tyr(269), which interact upstream and downstream of the abasic site, respectively, are involved in recognition and binding of abasic site-containing double stranded DNA. However, the two residues are not equivalent, as their effects are differentiated by changes in salt concentration. In sharp contrast, Tyr(171) is directly involved in catalysis as well as binding. Y171F, Y171H, and Y171A all show decreased catalytic efficiencies 25,000-50,000-fold from the WT enzyme. Both imidazole and basic pH markedly stimulate the WT enzyme. Imidazole stimulates Tyr(171) mutant enzymes when tyrosine is also present but basic pH eliminates remaining mutant activity. These results underscore the importance of tyrosines in AP endo catalysis. They render the current hypotheses regarding enzyme action unlikely and allow us to consider the possibility that the phenolate of Tyr(171) is the nucleophile that attacks the scissile phosphate.

Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography.

Purification of enveloped viruses such as live flavivirus vaccine candidates poses a challenge as one must retain viral infectivity to preserve immunogenicity. Here we describe a laboratory-scale purification procedure for two replication defective (single-cycle) flavivirus variants for use in a pre-clinical setting. The two step purification scheme based on hollow fiber tangential flow filtration (TFF) followed by anion exchange chromatography using convective interaction media (CIM(®)) monoliths results in a ∼60% recovery of infectious virus titer and can be used to prepare nearly homogenous, highly purified vaccine viruses with titers as high as 1×10(9) focus forming units per mL. Flavivirus virions prepared by this method are 2 and 3 orders of magnitude more pure with respect to dsDNA and BHK host cell proteins, respectively, as compared to the raw feed stream.

A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model.

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and B in vitro and because of their in vivo efficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains of C. difficile toxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

High-purity preparation of HSV-2 vaccine candidate ACAM529 is immunogenic and efficacious in vivo.

Genital herpes is a sexually transmitted infection (STI) caused by herpes simplex virus 2 (HSV-2) and to a lesser extent herpes simplex virus 1 (HSV-1). Infection by HSV-2 is life-long and is associated with significant cost to healthcare systems and social stigma despite the highly prevalent nature of the disease. For instance, the proportion of HSV-2 seropositive to seronegative adults is approximately 1 in 5 in the US and greater than 4 in 5 in some areas of sub-Saharan Africa. The replication-defective vaccine strain virus dl5-29 was re-derived using cells appropriate for GMP manufacturing and renamed ACAM529. Immunization with dl5-29 was previously reported to be protective both in mice and in guinea pigs, however these studies were performed with vaccine that was purified using methods that cannot be scaled for manufacturing of clinical material. Here we describe methods which serve as a major step towards preparation of ACAM529 which may be suitable for testing in humans. ACAM529 can be harvested from infected cell culture of the trans-complementing cell line AV529 clone 19 (AV529-19) without mechanical cell disruption. ACAM529 may then be purified with respect to host cell DNA and proteins by a novel purification scheme, which includes a combination of endonuclease treatment, depth filtration, anion-exchange chromatography and ultrafiltration/diafiltration (UF/DF). The resultant virus retains infectivity and is ∼ 200-fold more pure with respect to host cell DNA and proteins than is ACAM529 purified by ultracentrifugation. Additionally, we describe a side-by-side comparison of chromatography-purified ACAM529 with sucrose cushion-purified ACAM529, which shows that both preparations are equally immunogenic and protective when tested in vivo.

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.