Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study.

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.

Evaluation of Chimpanzee Adenovirus and MVA Expressing TRAP and CSP from Plasmodium cynomolgi to Prevent Malaria Relapse in Nonhuman Primates.

Plasmodium vivax is the world's most widely distributed human malaria parasite, with over 2.8 billion people at risk in Asia, the Americas, and Africa. The 80-90% new P. vivax malaria infections are due to relapses which suggest that a vaccine with high efficacy against relapses by prevention of hypnozoite formation could lead to a significant reduction in the prevalence of P. vivax infections. Here, we describe the development of new recombinant ChAdOx1 and MVA vectors expressing P. cynomolgi Thrombospondin Related Adhesive Protein (PcTRAP) and the circumsporozoite protein (PcCSP). Both were shown to be immunogenic in mice prior to their assessment in rhesus macaques. We confirmed good vaccine-induced humoral and cellular responses after prime-boost vaccination in rhesus macaques prior to sporozoite challenge. Results indicate that there were no significant differences between mock-control and vaccinated animals after challenge, in terms of protective efficacy measured as the time taken to 1st patency, or as number of relapses. This suggests that under the conditions tested, the vaccination with PcTRAP and PcCSP using ChAdOx1 or MVA vaccine platforms do not protect against pre-erythrocytic malaria or relapses despite good immunogenicity induced by the viral-vectored vaccines.

Chemoprophylaxis with sporozoite immunization in P. knowlesi rhesus monkeys confers protection and elicits sporozoite-specific memory T cells in the liver.

Whole malaria sporozoite vaccine regimens are promising new strategies, and some candidates have demonstrated high rates of durable clinical protection associated with memory T cell responses. Little is known about the anatomical distribution of memory T cells following whole sporozoite vaccines, and immunization of nonhuman primates can be used as a relevant model for humans. We conducted a chemoprophylaxis with sporozoite (CPS) immunization in P. knowlesi rhesus monkeys and challenged via mosquito bites. Half of CPS immunized animals developed complete protection, with a marked delay in parasitemia demonstrated in the other half. Antibody responses to whole sporozoites, CSP, and AMA1, but not CelTOS were detected. Peripheral blood T cell responses to whole sporozoites, but not CSP and AMA1 peptides were observed. Unlike peripheral blood, there was a high frequency of sporozoite-specific memory T cells observed in the liver and bone marrow. Interestingly, sporozoite-specific CD4+ and CD8+ memory T cells in the liver highly expressed chemokine receptors CCR5 and CXCR6, both of which are known for liver sinusoid homing. The majority of liver sporozoite-specific memory T cells expressed CD69, a phenotypic marker of tissue-resident memory (TRM) cells, which are well positioned to rapidly control liver-stage infection. Vaccine strategies that aim to elicit large number of liver TRM cells may efficiently increase the efficacy and durability of response against pre-erythrocytic parasites.

Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits.

One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood-stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 contains up to six recognition sites for N-linked glycosylation, a post-translational modification that is absent in P. falciparum. To prevent any potential negative impact of N-glycosylation, the recognition sites have been knocked out in most PfAMA1 variants expressed in eukaryotic hosts. However, N-linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 variants accumulated to high levels (>510 µg/g fresh leaf weight) after transient expression, and high-mannose-type N-glycans were confirmed for the glycosylated variant. No significant differences between the N. benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation-sensitive ligand-binding studies. Specific titres of >2 × 10(6) were induced in rabbits, and strong reactivity with P. falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC50 values of ~35 µg/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N-glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates.

Development and evaluation of a multiplex screening assay for Plasmodium falciparum exposure.

Transfusion transmitted malaria (TTM) in non-endemic countries is reduced by questioning blood donors and screening of donated blood. Conventional screening is performed by Indirect Fluorescence Antibody Test (IFAT). This method is manual and difficult to standardize. Here we study the diagnostic performance of a multiplex assay for detection of antibodies against Plasmodium falciparum in donor blood using IFAT as a comparator. A multiplex assay (MPA) containing the antigens GLURP-R0, GLURP-R2, MSP3, MSP1 hybrid and AMA1 was constructed using xMAP® technology. A discrimination index for exposure to P. falciparum malaria was calculated by comparing travelers with clinical malaria (n=52) and non-exposed blood donors (n=119). The index was evaluated on blood donors with suspected malaria exposure (n=249) and compared to the diagnostic performance of IFAT. At a specificity of 95.8 %, the MPA discrimination index exhibited a diagnostic sensitivity of 90.4 % in travelers hospitalized with malaria. Percent agreement with IFAT was 92.3 %. Screening plasma from blood donors with suspected malaria exposure, we found 4.8 % to be positive by IFAT and 5.2 % by MPA with an agreement of 93.2 %. The calculated index from the MPA exhibits similar diagnostic performance as IFAT for detection of P. falciparum malaria. Combining the antibody response against multiple antigens in a discrimination index increased the sensitivity of the MPA and reduced the readout to a single value.

Transgene optimization, immunogenicity and in vitro efficacy of viral vectored vaccines expressing two alleles of Plasmodium falciparum AMA1.

BACKGROUND: Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood-stage malaria, although to date numerous clinical trials using mainly protein-in-adjuvant vaccines have shown limited success. Here we describe the pre-clinical development and optimization of recombinant human and simian adenoviral (AdHu5 and ChAd63) and orthopoxviral (MVA) vectors encoding transgene inserts for Plasmodium falciparum AMA1 (PfAMA1). METHODOLOGY/PRINCIPAL FINDINGS: AdHu5-MVA prime-boost vaccination in mice and rabbits using these vectors encoding the 3D7 allele of PfAMA1 induced cellular immune responses as well as high-titer antibodies that showed growth inhibitory activity (GIA) against the homologous but not heterologous parasite strains. In an effort to overcome the issues of PfAMA1 antigenic polymorphism and pre-existing immunity to AdHu5, a simian adenoviral (ChAd63) vector and MVA encoding two alleles of PfAMA1 were developed. This antigen, composed of the 3D7 and FVO alleles of PfAMA1 fused in tandem and with expression driven by a single promoter, was optimized for antigen secretion and transmembrane expression. These bi-allelic PfAMA1 vaccines, when administered to mice and rabbits, demonstrated comparable immunogenicity to the mono-allelic vaccines and purified serum IgG now showed GIA against the two divergent strains of P. falciparum encoded in the vaccine. CD8(+) and CD4(+) T cell responses against epitopes that were both common and unique to the two alleles of PfAMA1 were also measured in mice. CONCLUSIONS/SIGNIFICANCE: Optimized transgene inserts encoding two divergent alleles of the same antigen can be successfully inserted into adeno- and pox-viral vaccine vectors. Adenovirus-MVA immunization leads to the induction of T cell responses common to both alleles, as well as functional antibody responses that are effective against both of the encoded strains of P. falciparum in vitro. These data support the further clinical development of these vaccine candidates in Phase I/IIa clinical trials.

Enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines.

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4(+) T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8(+) T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8(+) and CD4(+) T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.

Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein.

BACKGROUND: Plasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1. METHODS: A recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes. RESULTS: Fourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure. CONCLUSIONS: This study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations.

AIDS-protective HLA-B*27/B*57 and chimpanzee MHC class I molecules target analogous conserved areas of HIV-1/SIVcpz.

In the absence of treatment, most HIV-1-infected humans develop AIDS. However, a minority are long-term nonprogressors, and resistance is associated with the presence of particular HLA-B*27/B*57 molecules. In contrast, most HIV-1-infected chimpanzees do not contract AIDS. In comparison with humans, chimpanzees experienced an ancient selective sweep affecting the MHC class I repertoire. We have determined the peptide-binding properties of frequent chimpanzee MHC class I molecules, and show that, like HLA-B*27/B*57, they target similar conserved areas of HIV-1/SIV(cpz). In addition, many animals appear to possess multiple molecules targeting various conserved areas of the HIV-1/SIV(cpz) Gag protein, a quantitative aspect of the immune response that may further minimize the chance of viral escape. The functional characteristics of the contemporary chimpanzee MHC repertoire suggest that the selective sweep was caused by a lentiviral pandemic.

Malaria vaccine-related benefits of a single protein comprising Plasmodium falciparum apical membrane antigen 1 domains I and II fused to a modified form of the 19-kilodalton C-terminal fragment of merozoite surface protein 1.

We show that the smallest module of Plasmodium falciparum AMA1 (PfAMA1) that can be expressed in the yeast Pichia pastoris while retaining the capacity to induce high levels of parasite-inhibitory antibodies comprises domains I and II. Based on this, two fusion proteins, differing in the order of the modules, were developed. Each comprised one module of PfAMA1 (FVO strain, amino acids [aa] 97 to 442) (module A) and one module of PfMSP1(19) (Wellcome strain, aa 1526 to 1621) (module Mm) in which a cystine had been removed to improve immune responses. Both fusion proteins retained the antigenicity of each component and yielded over 30 mg/liter purified protein under fed-batch fermentation. Rabbits immunized with purified fusion proteins MmA and AMm had up to eightfold-higher immune responses to MSP1(19) than those of rabbits immunized with module Mm alone or Mm mixed with module A. In terms of parasite growth inhibition, fusion did not diminish the induction of inhibitory antibodies compared with immunization with module A alone or module A mixed with module Mm, and fusion outperformed antibodies induced by immunization with module M or Mm alone. When tested against parasites expressing AMA1 heterologous to the immunogen, antibodies to the fusion proteins inhibited parasite growth to a greater extent than did antibodies either to the individual antigens or to the mixture. These results suggest that compared with the individual modules delivered separately or as a mixture, fusion proteins containing these two modules offer the potential for significant vaccine-related advantages in terms of ease of production, immunogenicity, and functionality.

Production of IL-1beta and IL-1Ra as risk factors for susceptibility and progression of relapse-onset multiple sclerosis.

Interleukin-1beta (IL-1beta) is present in multiple sclerosis (MS) lesions. Interleukin-1 receptor antagonist (IL-1Ra) moderates the induction of experimental autoimmune encephalomyelitis (EAE). Here, we show that families that are characterized by high IL-1beta over IL-1Ra production ratio are at 2.2-fold (95% CI, 1.0-4.8; p=0.05) increased risk to have a patient relative with relapse-onset MS than families with a low ratio. It is also related to the reduction of volumetric magnetization transfer ratio (MTR) histogram height, a measure of parenchymal integrity (p=0.04). Those families who combine a high IL-1beta over IL-1Ra ratio with a high tumor necrosis factor (TNF) over IL-10 production ratio have a 6.2-fold (95% CI, 1.8-21; p=0.002) increased risk. Innate production of IL-1beta and IL-1Ra is not related to the outcome of primary progressive MS. Taq1 polymorphism in the IL-1beta gene and the variable number of tandem repeats (VNTR) polymorphism of 86-base pairs within the IL-1Ra gene cannot explain these findings.