Conserved Influenza Hemagglutinin, Neuraminidase and Matrix Peptides Adjuvanted with ALFQ Induce Broadly Neutralizing Antibodies

A universal influenza candidate vaccine that targets multiple conserved influenza virus epitopes from hemagglutinin (HA), neuraminidase (NA) and matrix (M2e) proteins was combined with the potent Army liposomal adjuvant (ALFQ) to promote induction of broad immunity to seasonal and pandemic influenza strains. The unconjugated and CRM-conjugated composite peptides formulated with ALFQ were highly immunogenic and induced both humoral and cellular immune responses in mice. Broadly reactive serum antibodies were induced across various IgG isotypes. Mice immunized with the unconjugated composite peptide developed antibody responses earlier than mice immunized with conjugated peptides, and the IgG antibodies were broadly reactive and neutralizing across Groups 1 and 2 influenza viruses. Multi-epitope unconjugated influenza composite peptides formulated with ALFQ provide a novel strategy for the development of a universal influenza vaccine. These synthetic peptide vaccines avoid the pitfalls of egg-produced influenza vaccines and production can be scaled up rapidly and economically.

Clinical and Prognostic Value of Immunogenetic Characteristics in Anti-LGI1 Encephalitis

ObjectiveAntibodies against leucine-rich glioma-inactivated 1 (LGI1-Abs) characterize a limbic encephalitis (LE) strongly associated with HLA-DRB1*07:01, although some patients lack LGI1-Abs in CSF or do not carry this allele. Whether they represent a different subtype of disease or have different prognoses is unclear.MethodsRetrospective analysis of clinical features, IgG isotypes, and outcome according to LGI1-Ab CSF positivity and DRB1*07:01 in a cohort of anti-LGI1 LE patients.ResultsPatients with LGI1-Abs detected in both CSF and serum (105/134, 78%) were compared with those who were CSF negative (29/134, 22%). Both groups had similar clinical features and serum levels, but CSF-positive patients had shorter diagnostic delay, more frequently hyponatremia, inflammatory CSF, and abnormal MRI (p < 0.05). Human leukocyte antigen (HLA) genotyping was performed in 72/134 (54%) patients and 63/72 (88%) carried DRB1*07:01. Noncarriers (9/72, 12%) were younger, more commonly women, and had less frequently psychiatric and frontal symptoms (p < 0.05). No difference in IgG isotypes according to CSF positivity or HLA was found (p > 0.05). HLA and IgG isotypes were not associated with poor outcome (mRS >2 at last follow-up) in univariate analyses; CSF positivity was only identified as a poor outcome predictor in the multivariate analysis including the complete follow-up, whereas age and female sex also remained when just the first year was considered.ConclusionsLE without CSF LGI1-Abs is clinically indistinguishable and likely reflects just a lesser LGI1-Ab production. HLA association is sex and age biased and presents clinical particularities, suggesting subtle differences in the immune response. Long-term outcome depends mostly on demographic characteristics and the intensity of the intrathecal synthesis.

Rapid structural discrimination of IgG antibodies by multicharge-state collision-induced unfolding

A simplified multicharge-state collision-induced unfolding (CIU) method was proposed for rapid differentiation of IgG isotypes that differ in terms of the numbers and patterns of disulfide bonds.

Serology confirms SARS-CoV-2 infection in PCR-negative children presenting with Paediatric Inflammatory Multi-System Syndrome

BackgroundDuring the COVID-19 outbreak, reports have surfaced of children who present with features of a multisystem inflammatory syndrome with overlapping features of Kawasaki disease and toxic shock syndrome – Paediatric Inflammatory Multisystem Syndrome-temporally associated with SARS-CoV-2 pandemic (PIMS-TS). Initial reports find that many of the children are PCR-negative for SARS-CoV-2, so it is difficult to confirm whether this syndrome is a late complication of viral infection in an age group largely spared the worst consequences of this infection, or if this syndrome reflects enhanced surveillance.MethodsChildren hospitalised for symptoms consistent with PIMS-TS between 28 April and 8 May 2020, and who were PCR-negative for SARS-CoV-2, were tested for antibodies to viral spike glycoprotein using an ELISA test.ResultsEight patients (age range 7–14 years, 63% male) fulfilled case-definition for PIMS-TS during the study period. Six of the eight patients required admission to intensive care. All patients exhibited significant IgG and IgA responses to viral spike glycoprotein. Further assessment showed that the IgG isotypes detected in children with PIMS-TS were of the IgGl and lgG3 subclasses, a distribution similar to that observed in samples from hospitalised adult COVID-19 patients. In contrast, lgG2 and lgG4 were not detected in children or adults. IgM was not detected in children, which contrasts with adult hospitalised adult COVID-19 patients of whom all had positive IgM responses.ConclusionsStrong IgG antibody responses can be detected in PCR-negative children with PIMS-TS. The low detection rate of IgM in these patients is consistent with infection having occurred weeks previously and that the syndrome onset occurs well after the control of SARS-CoV-2 viral load. This implies that the disease is largely immune-mediated. Lastly, this indicates that serology can be an appropriate diagnostic tool in select patient groups.

Murine Cross-Reactive Nonneutralizing Polyclonal IgG1 Antibodies Induced by Influenza Vaccine Inhibit the Cross-Protective Effect of IgG2 against Heterologous Virus in Mice

ABSTRACT Annual vaccination against influenza viruses is the most reliable and efficient way to prevent and control annual epidemics and protect from severe influenza disease. However, current split influenza vaccines are generally not effective against antigenically mismatched (heterologous) strains. To broaden the protective spectrum of influenza vaccines, adjuvants that can induce cross-reactive antibodies with cross-protection via Fc-mediated effector functions are urgently sought. Although IgG2 antibodies are generally more efficient than IgG1 antibodies in Fc-mediated effector functions, it is not yet clear which IgG isotypes show superior cross-protection against heterologous strains. It also remains unclear whether these IgG isotypes interfere with each other’s protective effects. Here, we found that influenza split vaccine adjuvanted with aluminum salts, which predominantly induce cross-reactive IgG1, did not confer cross-protection against heterologous virus challenge in mice. In contrast, split vaccine adjuvanted with CpG oligodeoxynucleotides, which predominantly induce cross-reactive IgG2, showed cross-protection through the interaction of cross-reactive nonneutralizing IgG2 and alveolar macrophages, indicating the importance of cross-reactive nonneutralizing IgG2 for cross-protection. Furthermore, by using serum samples from immunized mice and isolated polyclonal antibodies, we show that vaccine-induced cross-reactive nonneutralizing IgG1 suppress the cross-protective effects of IgG2 by competitively inhibiting the binding of IgG2 to virus. Thus, we demonstrate the new concept that cross-reactive IgG1 may interfere with the potential for cross-protection of influenza vaccine. We propose that adjuvants that selectively induce virus-specific IgG2 in mice, such as CpG oligodeoxynucleotides, are optimal for heterologous protection. IMPORTANCE Current influenza vaccines are generally effective against highly similar virus strains by inducing neutralizing antibodies. However, these antibodies fail to neutralize antigenically mismatched (heterologous) strains and therefore provide limited protection against them. Efforts are being made to develop vaccines with cross-protective ability that would protect broadly against heterologous strains, because the mismatch between predicted and epidemic strains cannot always be avoided, resulting in low vaccine efficacy. Here, we show that nonneutralizing IgG2 antibodies induced by an optimal adjuvant play a crucial role in cross-protection against heterologous virus challenge in mice. Furthermore, nonneutralizing polyclonal IgG1 suppressed the cross-protective effects of nonneutralizing polyclonal IgG2 by competitively blocking the binding of IgG2 to its antigen. These data shed new light on the importance of IgG isotypes and the selection of appropriate adjuvants for the development of universal influenza vaccines. Furthermore, our findings are applicable to the rational design of vaccines against other pathogens.

Antenatal Influenza A-Specific IgA, IgM, and IgG Antibodies in Mother’s Own Breast Milk and Donor Breast Milk, and Gastric Contents and Stools from Preterm Infants

Antenatal milk anti-influenza antibodies may provide additional protection to newborns until they are able to produce their own antibodies. To evaluate the relative abundance of milk, we studied the antibodies specific to influenza A in feeds and gastric contents and stools from preterm infants fed mother’s own breast milk (MBM) and donor breast milk (DBM). Feed (MBM or DBM) and gastric contents (MBM or DBM at 1 h post-ingestion) and stool samples (MBM/DBM at 24 h post-ingestion) were collected, respectively, from 20 preterm (26–36 weeks gestational age) mother-infant pairs at 8–9 days and 21–22 days of postnatal age. Samples were analyzed via ELISA for anti-H1N1 hemagglutinin (anti-H1N1 HA) and anti-H3N2 neuraminidase (anti-H3N2 NA) specificity across immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) isotypes. The relative abundance of influenza A-specific IgA in feeds and gastric contents were higher in MBM than DBM at 8–9 days of postnatal age but did not differ at 21–22 days. Anti-influenza A-specific IgM was higher in MBM than in DBM at both postnatal times in feed and gastric samples. At both postnatal times, anti-influenza A-specific IgG was higher in MBM than DBM but did not differ in gastric contents. Gastric digestion reduced anti-H3N2 NA IgG from MBM at 21–22 days and from DBM at 8–9 days of lactation, whereas other anti-influenza A antibodies were not digested at either postnatal times. Supplementation of anti-influenza A-specific antibodies in DBM may help reduce the risk of influenza virus infection. However, the effective antibody dose required to induce a significant protective effect remains unknown.

Identification and Characterization of Novel Fc-Binding Heptapeptides from Experiments and Simulations

Purification of biologically-derived therapeutics is a major cost contributor to the production of this rapidly growing class of pharmaceuticals. Monoclonal antibodies comprise a large percentage of these products, therefore new antibody purification tools are needed. Small peptides, as opposed to traditional antibody affinity ligands such as Protein A, may have advantages in stability and production costs. Multiple heptapeptides that demonstrate Fc binding behavior that have been identified from a combinatorial peptide library using M13 phage display are presented herein. Seven unique peptide sequences of diverse hydrophobicity and charge were identified. All seven peptides showed strong binding to the four major human IgG isotypes, human IgM, as well as binding to canine, rat, and mouse IgG. These seven peptides were also shown to bind human IgG4 from DMEM cell culture media with 5% FCS and 5 g/L ovalbumin present. These peptides may be useful as surface ligands for antibody detection and purification purposes. Molecular docking and classical molecular dynamics (MD) simulations were conducted to elucidate the mechanisms and energetics for the binding of these peptides to the Fc region. The binding site was found to be located between the two glycan chains inside the Fc fragment. Both hydrogen bonding and hydrophobic interactions were found to be crucial for the binding interactions. Excellent agreement for the binding strength was obtained between experimental results and simulations.

The Development and Study of Recombinant Immunoglobulin A to Hemagglutinins of the Influenza Virus

We obtained recombinant variants of human antibody FI6 broadly specific to hemagglutinins of the influenza A virus. On the basis of a bi-promoter (CMV, hEF1-HTLV) vector, we developed genetic constructs for the expression of the heavy and light chains of the immunoglobulins of IgA1-, IgA2m1-, and IgG-isotypes. Following transfection and selection, stable Chinese hamster ovary (CHO) cell lines were produced. The antibodies of IgA1-, IgA2m1-, and IgG-isotypes were purified from culture media. We performed an immunochemical characterization and studied their interactions with influenza A strains of the H1N1- and H3N2-subtypes. It was shown that recombinant FI6 variants of the IgA-isotype retain the properties of the parental IgG antibody to demonstrate specificity to all the strains tested. The strongest binding was observed for the H1N1 subtype, which belongs to hemagglutinins of phylogenetic group I.

Identification and Characterization of Novel Fc-Binding Heptapeptides from Experiments and Simulations

Purification of biologically-derived therapeutics is a major cost contributor to the production of this rapidly growing class of pharmaceuticals. Monoclonal antibodies comprise a large percentage of these products therefore new antibody purification tools are needed. Small peptides, as opposed to traditional antibody affinity ligands such as Protein A, may have advantages in stability and production costs. Multiple heptapeptides that demonstrate Fc binding behavior that have been identified from a combinatorial peptide library using M13 Phage Display are presented herein. Seven unique peptide sequences of diverse hydrophobicity and charge were identified. All seven peptides showed strong binding to the four major human IgG isotypes, human IgM, as well as binding to canine, rat, and mouse IgG. These seven peptides were also shown to bind human IgG4 from DMEM cell culture media with 5% FCS and 5 g/L ovalbumin present. These peptides may be useful as surface ligands for antibody detection and purification purposes. Molecular docking and classical molecular dynamics (MD) simulations were conducted to elucidate the mechanisms and energetics for the binding of these peptides to the Fc region. The binding site was found to be located between the two glycan chains inside the Fc fragment. Both hydrogen bonding and hydrophobic interactions were found to be crucial for the binding interactions. Excellent agreement for the binding strength was obtained between experimental results and simulations.