A prime-boost concept using a T-cell epitope-driven DNA vaccine followed by a whole virus vaccine effectively protected pigs in the pandemic H1N1 pig challenge model

Influenza A virus (IAV) vaccines in pigs generally provide homosubtypic protection but fail to prevent heterologous infections. In this pilot study, the efficacy of an intradermal pDNA vaccine composed of conserved SLA class I and class II T cell epitopes (EPITOPE) against a homosubtypic challenge was compared to an intramuscular commercial inactivated whole virus vaccine (INACT) and a heterologous prime boost approach using both vaccines. Thirty-nine IAV-free, 3-week-old pigs were randomly assigned to one of five groups including NEG-CONTROL (unvaccinated, sham-challenged), INACT-INACT-IAV (vaccinated with FluSure XP® at 4 and 7 weeks, pH1N1 challenged), EPITOPE-INACT-IAV (vaccinated with PigMatrix EDV at 4 and FluSure XP® at 7 weeks, pH1N1 challenged), EPITOPE-EPITOPE-IAV (vaccinated with PigMatrix EDV at 4 and 7 weeks, pH1N1 challenged), and a POS-CONTROL group (unvaccinated, pH1N1 challenged). The challenge was done at 9 weeks of age and pigs were necropsied at day post challenge (dpc) 5. At the time of challenge, all INACT-INACT-IAV pigs, and by dpc 5 all EPITOPE-INACT-IAV pigs were IAV seropositive. IFNγ secreting cells, recognizing vaccine epitope-specific peptides and pH1N1 challenge virus were highest in the EPITOPE-INACT-IAV pigs at challenge. Macroscopic lung lesion scores were reduced in all EPITOPE-INACT-IAV pigs while INACT-INACT-IAV pigs exhibited a bimodal distribution of low and high scores akin to naïve challenged animals. No IAV antigen in lung tissues was detected at necropsy in the EPITOPE-INACT-IAV group, which was similar to naïve unchallenged pigs and different from all other challenged groups. Results suggest that the heterologous prime boost approach using an epitope-driven DNA vaccine followed by an inactivated vaccine was effective against a homosubtypic challenge, and further exploration of this vaccine approach as a practical control measure against heterosubtypic IAV infections is warranted.     

Modular epitope binding predicts influenza quasispecies dominance and vaccine effectiveness: Application to 2018/19 season

The modular binding sites on the influenza A(H3N2) hemagglutinin protein are under significant pressure to acquire mutations in order to evade human antibody recognition. Analysis of these hemagglutinin epitopes in the strains circulating during 2017/18 and early 2018/19 identified the emergence of a new antigenic cluster that has grown from 4% of circulating strains to 11%. We regressed our module-based antigenic distance, p_epitope, with A(H3N2) vaccine effectiveness from recent studies conducted by the US Centers for Disease Control and Prevention (r² = 0.92), and we used this to estimate that the 2018/19 vaccines will protect against most circulating A(H3N2) strains. The pEpitope model is useful for A(H3N2) influenza vaccine virus selection and development, and it has the potential to aid national or regional regulatory authorities in making geographically localized decisions.     

Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263)

BackgroundFor the 2017–18 influenza season, A/Saitama/103/2014 (CEXP-002) (Saitama strain) was antigenically more similar to prior circulating strains than A/Hong Kong/4801/2014 (X-263) (Hong Kong strain) in a ferret model and was selected as the A(H3N2) vaccine virus strain in Japan. However, the Saitama strain grew poorly, and the Japanese government switched to the Hong Kong strain, raising public concerns of poor effectiveness. To enhance understanding of the correlation between antigenicity in experimental models and immunogenicity, as a surrogate measure of vaccine effectiveness, in the human population, we compared the immunogenicity of specially-prepared single dose monovalent influenza A(H3N2) vaccines containing the Saitama or the Hong Kong strain.MethodsA randomized controlled trial of 100 healthy adults aged 20–64 years (n = 50/group) was conducted. Virus neutralization assay was performed on sera from days 0 (pre-vaccination) and 21 (post-vaccination). Geometric mean titer (GMT), mean fold rise (MFR), seroconversion proportion (SCP), and seroprotection proportion (SPP) were calculated for vaccine strains and a representative circulating A(H3N2) virus strain (A/Osaka/188/2017).ResultsFor the Hong Kong strain, post-vaccination GMT was significantly higher in the Hong Kong vaccine recipients (1:546 vs 1:260, p < 0.01), but MFR, SCP, and SPP were similar for both vaccine groups. For the Saitama strain, post-vaccination GMT (1:116 vs 1:61, p = 0.01) and SPP (86% vs 68%, p = 0.03) were significantly higher in the Hong Kong vaccine recipients, but MFR and SCP were similar for both vaccine groups. Against A/Osaka/188/2017, post-vaccination GMT and MFR were similar in both vaccine groups, but SCP (32% vs 4%, p < 0.01) and SPP (28% vs. 6%, p < 0.01) were significantly higher in the Hong Kong vaccine recipients.ConclusionThe Hong Kong vaccine induced better or equivalent immunogenicity in comparison to the Saitama vaccine. Our trial showed that antigenic similarity in experimental models does not necessarily correlate with immunogenicity in the human population.Clinical trial registration: UMIN000029293.     

Vaccination against cocaine using a modifiable dendrimer nanoparticle platform

Pharmacological therapies for the treatment of cocaine addiction have had disappointing efficacy, and the lack of recent developments in the clinical care of cocaine-addicted patients indicates a need for novel treatment strategies. Recent studies have shown that vaccination against cocaine to elicit production of antibodies that reduce concentrations of free drug in the blood is a promising method to protect against the effects of cocaine and reduce rates of relapse. However, the poorly immunogenic nature of cocaine remains a major hurdle to active immunization. Therefore, we hypothesized that strategies to increase targeted exposure of cocaine to the immune system may produce a more effective vaccine. To specifically direct an immune response against cocaine, in the present study we have conjugated a cocaine analog to a dendrimer-based nanoparticle carrier with MHC II-binding moieties that previously has been shown to activate antigen-presenting cells necessary for antibody production. This strategy produced a rapid, prolonged, and high affinity anti-cocaine antibody response without the need for an adjuvant. Surprisingly, additional evaluation using multiple adjuvant formulations in two strains of inbred mice found adjuvants were either functionally redundant or deleterious in the vaccination against cocaine using this platform. The use of conditioned place preference in rats after administration of this vaccine provided proof of concept for the ability of this vaccine to diminish cocaine reward. Together these data demonstrate the intrinsic efficacy of an immune-targeting dendrimer-based cocaine vaccine, with a vast potential for design of future vaccines against other poorly immunogenic antigens by substitution of the conjugated cargo.     

Endpoint and epitope-specific antibody responses as correlates of vaccine-mediated protection of mice against ricin toxin

The successful licensure of vaccines for biodefense is contingent upon the availability of well-established correlates of protection (CoP) in at least two animal species that can be applied to humans, without the need to assess efficacy in the clinic. In this report we describe a multivariate model that combines pre-challenge serum antibody endpoint titers (EPT) and values derived from an epitope profiling immune-competition capture (EPICC) assay as a predictor in mice of vaccine-mediated immunity against ricin toxin (RT), a Category B biothreat. EPICC is a modified competition ELISA in which serum samples from vaccinated mice were assessed for their ability to inhibit the capture of soluble, biotinylated (b)-RT by a panel of immobilized monoclonal antibodies (mAbs) directed against four immunodominant toxin-neutralizing regions on the enzymatic A chain (RTA) of RT. In a test cohort of mice (n = 40) vaccinated with suboptimal doses of the RTA subunit vaccine, RiVax®, we identified two mAbs, PB10 and SyH7, which had EPICC inhibition values in pre-challenge serum samples that correlated with survival following a challenge with 5 × LD₅₀ of RT administered by intraperitoneal (IP) injection. Analysis of a larger cohort of mice (n = 645) revealed that a multivariate model combining endpoint titers and EPICC values for PB10 and SyH7 as predictive variables had significantly higher statistical power than any one of the independent variables alone. Establishing the correlates of vaccine-mediated protection in mice represents an important steppingstone in the development of RiVax® as a medical countermeasure under the United States Food and Drug Administration’s “Animal Rule.”     

Identification of a Major Epitope Recognized by PLA2R Autoantibodies in Primary Membranous Nephropathy

Phospholipase A₂ receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD). However, in nondenaturing conditions the epitope was protected against reduction in larger fragments, including the full-length extracellular region of PLA2R. To determine the composition of the epitope, we isolated immunoreactive tryptic fragments by Western blotting and analyzed them by mass spectrometry. The identified peptides were tested as inhibitors of autoantibody binding to PLA2R by surface plasmon resonance. Two peptides from the ricin domain showed strong inhibition, with a longer sequence covering both peptides (31-mer) producing 85% inhibition of autoantibody binding to PLA2R. Anti-PLA2R antibody directly bound this 31-mer peptide under nondenaturing conditions and binding was sensitive to reduction. Analysis of PLA2R and the PLA2R-anti-PLA2R complex using electron microscopy and homology-based representations allowed us to generate a structural model of this major epitope and its antibody binding site, which is independent of pH-induced conformational change in PLA2R. Identification of this major PLA2R epitope will enable further therapeutic advances for patients with idiopathic membranous nephropathy, including antibody inhibition therapy and immunoadsorption of circulating autoantibodies.     

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5–10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular condition that afflicts as many as 1 of 350 males and 420 females over the age of 18 (1). In ALS, degeneration of upper and lower motor neurons causes progressive muscle paralysis and spasticity, affecting mobility, speech, swallowing, and respiration (2). Half of affected individuals die within 3 y, and less than 20% survive for more than 5 y (3); 90–95% of ALS cases are sporadic (SALS) in which some apparently facilitating gene mutations, such as repeat expansions in the gene that encodes ataxin-2 (4), have been identified. The remaining 5–10% of ALS cases are familial (FALS) and predominantly associated with Mendelian-inherited mutations in the genes encoding Cu/Zn superoxide dismutase (SOD1), TAR-DNA–binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/TLS), C9ORF72, and other genes (reviewed in ref. 3).Despite the profusion of functionally diverse genes implicated in FALS and SALS, clinical and pathological similarities between all forms of ALS suggest the existence of a common pathogenic pathway that could be united by a single gene/protein (5). One of the mechanisms by which a mutant or wild-type (WT) protein can dominate pathogenesis of phenotypically diverse diseases is by propagated protein misfolding, such as that underpinning the prion diseases, which has been increasingly implicated in other neurodegenerative and systemic disorders (6, 7). A role for propagated protein misfolding in ALS is supported by the prion-like spatiotemporal progression of disease through the neuroaxis (8, 9). However, given the disparity in protein inclusion pathology between subtypes of ALS, a single unifying prion-like protein that could explain such a progression remains obscure.Whereas it is generally accepted SOD1 is not found in large perikaryal cytoplasmic inclusions outside of SOD1 FALS cases, misfolded SOD1 has been increasingly identified in SALS and non-SOD1 FALS (5, 10, 11). Indeed, we have reported that misfolded human wild-type SOD1 (HuWtSOD1) can be detected by spinal cord immunohistochemistry (IHC) in FALS secondary to FUS mutation, and in SALS patients with cytosolic WT TDP-43 accumulation (11). Moreover, in cell models, overexpression of WTTDP-43, or expression of mutant FUS or TDP-43, is associated with HuWtSOD1 misfolding (11). Collectively, these data are consistent with SOD1 being a molecular common denominator for all types of ALS. Furthermore, prion-like activity has been described for the cell-to-cell transmission of misfolding of mutant SOD1 (12), and we have reported that mutant SOD1 can confer its misfold on HuWtSOD1 (13). However, mutant SOD1 cannot explain propagation in SALS.To test if HuWtSOD1 participates in cell-to-cell transmission of protein misfolding, we make use of previously developed mouse mAb probes for misfolded/oxidized SOD1, recognizing either full-length human mutant or WT SOD1, generated against regions that are antibody-inaccessible in natively folded SOD1 (13–15). Misfolded SOD1 mAbs used in this work are 10E11C11 and 3H1, directed against an unstructured electrostatic loop [disease-specific epitope-2 (DSE2)], and 10C12, directed against a C-terminal dimer interface peptide in which the cysteine at position 146 is substituted by a cysteic acid residue to mimic oxidation of this residue (DSE1a) (13). The use of such antibody probes have enabled us to unambiguously determine the role of misfolded mutant G127X in the induced misfolding of HuWtSOD1, which upon misfolding acquires a marked increase in sensitivity to protease digestion, consistent with global loosening of structure (13). The finding that misfolded endogenous HuWtSOD1 was observed long after transfected G127X-SOD1 was degraded suggested that HuWtSOD1, once misfolded, is capable of triggering an intracellular propagated misfolding reaction (13). We now report for the first time that misfolded HuWtSOD1 can transit cell to cell both via exosomes, and release of protein aggregates and subsequent uptake in neuronal cells. In addition, misfolded HuWtSOD1 can sustain intercellular propagated misfolding in vitro and is detectable in the spinal cord of all ALS patients tested, regardless of the genetic etiology of the disease. Collectively, these data indicate that HuWtSOD1 is competent to participate in propagated misfolding, suggesting a common pathogenic mechanism linking FALS and SALS.     

Analysis of density and epitopes of D antigen on the surface of erythrocytes from DEL phenotypic individuals carrying the RHD1227A allele

Background

The characteristics of the D antigen are important as they influence the immunogenicity of D variant cells. Several studies on antigenic sites have been reported in normal D positive, weak D and partial D cases, including a comprehensive analysis of DEL types in Caucasians. The aim of this study was to assess D antigen density and epitopes on the erythrocyte surface of Asian type DEL phenotypic individuals carrying the RHD1227A allele in the Chinese population.

Materials and methods

A total of 154 DEL phenotypic individuals carrying the RHD1227A allele were identified through adsorption and elution tests and polymerase chain reaction analysis with sequence-specific primers in the Chinese population. D antigen density on the erythrocyte surface of these individuals was detected using a flow cytometric method. An erythrocyte sample with known D antigen density was used as a standard. Blood samples from D-negative and D-positive individuals were used as controls. In addition, D antigen epitopes on the erythrocyte surface of DEL individuals carrying the RHD1227A allele were investigated with 18 monoclonal anti-D antibodies specific for different D antigen epitopes.

Results

The means of the median fluorescence intensity of D antigen on the erythrocyte membrane surface of D-negative, D-positive and DEL individuals were 2.14±0.25, 193.61±11.43 and 2.45±0.82, respectively. The DEL samples were estimated to have approximately 22 D antigens per cell. The samples from all 154 DEL individuals reacted positively with 18 monoclonal anti-D antibodies specific for different D antigen epitopes.

Discussion

In this study, D antigen density on the erythrocyte surface of DEL individuals carrying the RHD1227A allele was extremely low, there being only very few antigenic molecules per cell, but the D antigen epitopes were grossly complete.     

Reduced expression of macrophage-associated antigens on alveolar mononuclear phagocytes from acquired immunodeficiency syndrome

Summary In this study we evaluated the phenotype of alveolar mononuclear phagocytes recovered from the bronchoalveolar lavage fluid of 24 patients with human immunodeficiency virus infection (AIDS-related complex 8 patients, AIDS 16 patients) and 8 healthy individuals by using a panel of monoclonal antibodies known to react with tissue macrophages, in combination with a flow cytometer. The results showed that 90% of patients with AIDS present a marked reduction in the expression of several antigenic determinants (in descreasing order: CD68, CD36, CR1, CD11c, HLA-DR). The levels of antigen expression by flow cytometry seem to decline with disease progression, showing the most dramatic perturbations in patients with full-blown AIDS associated with pulmonary infections (especiallyPneumocystis carinii pneumonia) and lower peripheral CD4 lymphocyte counts. In contrast, patients with AIDS-related complex or AIDS without histological or cultural evidence of pulmonary involvement showed, respectively, only minimal or medium antigenic decreases. However, only a minor proportion (16%, 20%, 20%, 25%, and 25% respectively) of human immunodeficiency virus infected patients (mostly with AIDS) had a significant reduction of the levels of CD4, CD14, CD45R, CD11b, and CD16 antigens in the alveolar macrophages. Since macrophages play a central role in the pathogenesis of AIDS, it may be postulated that the loss of various phenotypic markers on alveolar mononuclear phagocytes (some of them known for their important immunoregulatory actions) could have an important part in the pathogenesis of human immunodeficiency virus induced immunosuppression, and thereby condition the abnormal susceptibility to pulmonary diseases typical of human immunodeficiency virus-infected patients.