Endpoint Titers Research Articles

Considerations for Endpoint Titer Determination in Syphilis Testing Using Newly Marketed, Automated Rapid Plasma Reagin Instruments.

Considerations for Endpoint Titer Determination in Syphilis Testing Using Newly Marketed, Automated Rapid Plasma Reagin Instruments.

A self-binding immune complex vaccine elicits strong neutralizing responses against herpes simplex virus in mice.

It has been known for over half a century that mixing an antigen with its cognate antibody in an immune complex (IC) can enhance antigen immunogenicity. However, many ICs produce inconsistent immune responses, and the use of ICs in the development new vaccines has been limited despite the otherwise widespread success of antibody-based therapeutics. To address this problem, we designed a self-binding recombinant immune complex (RIC) vaccine which mimics the larger ICs generated during natural infection. In this study, we created two novel vaccine candidates: 1) a traditional IC targeting herpes simplex virus 2 (HSV-2) by mixing glycoprotein D (gD) with a neutralizing antibody (gD-IC); and 2) an RIC consisting of gD fused to an immunoglobulin heavy chain and then tagged with its own binding site, allowing self-binding (gD-RIC). We characterized the complex size and immune receptor binding characteristics in vitro for each preparation. Then, the in vivo immunogenicity and virus neutralization of each vaccine were compared in mice. gD-RIC formed larger complexes which enhanced C1q receptor binding 25-fold compared to gD-IC. After immunization of mice, gD-RIC elicited up to 1,000-fold higher gD-specific antibody titers compared to traditional IC, reaching endpoint titers of 1:500,000 after two doses without adjuvant. The RIC construct also elicited stronger virus-specific neutralization against HSV-2, as well as stronger cross-neutralization against HSV-1, although the proportion of neutralizing antibodies to total antibodies was somewhat reduced in the RIC group. This work demonstrates that the RIC system overcomes many of the pitfalls of traditional IC, providing potent immune responses against HSV-2 gD. Based on these findings, further improvements to the RIC system are discussed. RIC have now been shown to be capable of inducing potent immune responses to a variety of viral antigens, underscoring their broad potential as a vaccine platform.

Combination adjuvants and antigens provide heterosubtypic protection against lethal influenza infection.

Abstract Influenza A virus (IAV) is a negative strand RNA virus that undergoes antigenic drift and shift, leading to serologically distinct strains that can cause tens of thousands of deaths annually in the US. The wide variety of hemagglutinin (HA) and neuraminidase (NA) proteins necessitates yearly reformulation of the vaccine to protect against novel seasonal and pandemic strains. Current IAV vaccines do not contain adjuvants, however, immune modulators such as pattern recognition receptor (PRR) agonists may enhance antigen presentation to T cells and provide broader protection against IAV strains that differ in HA and NA outer coat proteins. We set out to investigate whether TLR4 (lipopolysaccharide) and TLR3 (poly IC) agonists in combination with HA (H3N2) and nucleoprotein (NP) or NP and matrix (M1) proteins could provide protection against lethal challenge with mismatched IAV strain H1N1. Mice given H3 and NP with LPS + pIC in a prime/boost strategy demonstrated 80% survival after heterosubtypic H1N1 challenge while H3 + NP in PBS showed 20% survival. Similar results were seen using LPS + pIC in combination with NP and M1 protein with 90% survival. High endpoint titers of anti-NP antibodies (that also bound H1N1 virus) correlated with heterosubtypic protection suggesting a role for non-neutralizing antibodies in promoting survival. In addition, LPS and pIC enhanced dendritic cell (DC) function in vitro and increased migratory monocyte DC in the lymph node in vivo, coupled with early upregulation of T cell marker CD69. This strategy may provide increased efficacy over current IAV vaccines by inducing non-neutralizing antibodies and enhanced T cell responses to internal proteins. Supported by Trudeau Institute and Clarkson University.

Diagnostic implications of MOG-IgG detection in sera and cerebrospinal fluids.

The spectrum of MOG-IgG-associated disease (MOGAD) includes optic neuritis (ON), myelitis (MY), acute disseminated encephalomyelitis (ADEM), brainstem encephalitis, cerebral cortical encephalitis (CE) and AQP4-IgG-negative neuromyelitis optica spectrum disorder (NMOSD). In MOGAD, MOG-IgG are usually detected in sera (MOG-IgGSERUM), but there have been some seronegative MOGAD cases with MOG-IgG in CSF (MOG-IgGCSF), and its diagnostic implications remains unclear. In this cross-sectional study, we identified patients with paired serum and CSF sent from all over Japan for testing MOG-IgG. Two investigators blinded to MOG-IgG status classified them into suspected MOGAD (ADEM, CE, NMOSD, ON, MY and Others) or not based on the current recommendations. The MOG-IgGSERUM and MOG-IgGCSF titres were assessed with serial 2-fold dilutions to determine end point titres [≥1:128 in serum and ≥1:1 (no dilution) in CSF were considered positive]. We analysed the relationship between MOG-IgGSERUM, MOG-IgGCSF and the phenotypes with multivariable regression. A total of 671 patients were tested [405 with suspected MOGAD, 99 with multiple sclerosis, 48 with AQP4-IgG-positive NMOSD and 119 with other neurological diseases (OND)] before treatment. In suspected MOGAD, 133 patients (33%) tested MOG-IgG-positive in serum and/or CSF; 94 (23%) double-positive (ADEM 36, CE 15, MY 8, NMOSD 9, ON 15 and Others 11); 17 (4.2%) serum-restricted-positive (ADEM 2, CE 0, MY 3, NMOSD 3, ON 5 and Others 4); and 22 (5.4%) CSF-restricted-positive (ADEM 3, CE 4, MY 6, NMOSD 2, ON 0 and Others 7). None of AQP4-IgG-positive NMOSD, multiple sclerosis or OND cases tested positive for MOG-IgGSERUM, but two with multiple sclerosis cases were MOG-IgGCSF-positive; the specificities of MOG-IgGSERUM and MOG-IgGCSF in suspected MOGAD were 100% [95% confidence interval (CI) 99-100%] and 99% (95% CI 97-100%), respectively. Unlike AQP4-IgG-positive NMOSD, the correlation between MOG-IgGSERUM and MOG-IgGCSF titres in MOGAD was weak. Multivariable regression analyses revealed MOG-IgGSERUM was associated with ON and ADEM, whereas MOG-IgGCSF was associated with ADEM and CE. The number needed to test for MOG-IgGCSF to diagnose one additional MOGAD case was 13.3 (14.3 for ADEM, 2 for CE, 19.5 for NMOSD, infinite for ON, 18.5 for MY and 6.1 for Others). In terms of MOG-IgGSERUM/CSF status, most cases were double-positive while including either serum-restricted (13%) or CSF-restricted (17%) cases. These statuses were independently associated with clinical phenotypes, especially in those with ON in serum and CE in CSF, suggesting pathophysiologic implications and the utility of preferential diagnostic testing. Further studies are warranted to deduce the clinical and pathological significance of compartmentalized MOG-IgG.

Precision and correlation of ED50 and endpoint titer method in measuring HPV vaccine immunogenicity

Cervical cancer, the second leading cause of cancer-related deaths among women, is caused by human papillomavirus (HPV), a sexually transmitted virus. Vaccination is an effective preventive measure against viral infections and subsequent development of cervical cancer. Enzyme-linked immunosorbent assay (ELISA) is commonly used to measure specific binding antibody titers and assess the immunogenicity of test vaccines in preclinical models or clinical volunteers. Two methods of deriving titers, the endpoint titer (ET) and the effective dilution producing a median maximal effective fold of dilution (ED50) with a cut-off value, are widely used. For HPV, a pseudovirion-based neutralization assay (PBNA) is used to measure functional antibody titers. The ELISA binding titers and functional PBNA titers were found to be well-correlated for all nine HPV types tested in the vaccine, consistent with previous studies on HPV 16/18. Comparing the PBNA results with the two titration methods, the ED50 method showed higher precision and a closer correlation with PBNA results, both for individual types and pooled data analysis for all nine types. When comparing the titration results of the ET method based on a cut-off value with the ED50 method using all the data points across the dilution series, the ED50 method demonstrated better precision and a stronger correlation with PBNA results.

Serological cross-reactivity between spotted fever and typhus groups of rickettsia infection in Japan

ObjectivesWe examined the frequency of cross-reactions to Rickettsia typhi in patients with Japanese spotted fever (JSF) and evaluated the differences between two rickettsiae using antibody endpoint titers. MethodsPatients’ immunoglobulin (Ig)M and IgG titers against Rickettsia japonica and Rickettsia typhi in two phases were measured using an indirect immunoperoxidase assay at two reference centers for rickettsiosis in Japan. Cross-reaction was defined as a higher titer against R. typhi in convalescent sera than in acute sera among patients fulfilling the criteria for JSF diagnosis. The frequencies of IgM and IgG were also evaluated. ResultsApproximately 20% of cases showed positive cross-reactions. A comparison of antibody titers revealed the difficulty in identifying some positive cases. ConclusionCross-reactions of 20% in serodiagnosis may lead to the misclassification of rickettsial diseases. However, with the exception of some cases, we were able to successfully differentiate JSF from murine typhus using each endpoint titer.

Flexibility of the Rotavirus NSP2 C-Terminal Region Supports Factory Formation via Liquid-Liquid Phase Separation.

Many viruses sequester the materials needed for their replication into discrete subcellular factories. For rotaviruses (RVs), these factories are called viroplasms, and they are formed in the host cell cytosol via the process of liquid-liquid phase separation (LLPS). The nonstructural protein 2 (NSP2) and its binding partner, nonstructural protein 5 (NSP5), are critical for viroplasm biogenesis. Yet it is not fully understood how NSP2 and NSP5 cooperate to form factories. The C-terminal region (CTR) of NSP2 (residues 291 to 317) is flexible, allowing it to participate in domain-swapping interactions that promote interoctamer interactions and, presumably, viroplasm formation. Molecular dynamics simulations showed that a lysine-to-glutamic acid change at position 294 (K294E) reduces NSP2 CTR flexibility in silico. To test the impact of reduced NSP2 CTR flexibility during infection, we engineered a mutant RV bearing this change (rRV-NSP2K294E). Single-cycle growth assays revealed a >1.2-log reduction in endpoint titers for rRV-NSP2K294E versus the wild-type control (rRV-WT). Using immunofluorescence assays, we found that rRV-NSP2K294E formed smaller, more numerous viroplasms than rRV-WT. Live-cell imaging experiments confirmed these results and revealed that rRV-NSP2K294E factories had delayed fusion kinetics. Moreover, NSP2K294E and several other CTR mutants formed fewer viroplasm-like structures in NSP5 coexpressing cells than did control NSP2WT. Finally, NSP2K294E exhibited defects in its capacity to induce LLPS droplet formation in vitro when incubated alongside NSP5. These results underscore the importance of NSP2 CTR flexibility in supporting the biogenesis of RV factories. IMPORTANCE Viruses often condense the materials needed for their replication into discrete intracellular factories. For rotaviruses, agents of severe gastroenteritis in children, factory formation is mediated in part by an octameric protein called NSP2. A flexible C-terminal region of NSP2 has been proposed to link several NSP2 octamers together, a feature that might be important for factory formation. Here, we created a change in NSP2 that reduced C-terminal flexibility and analyzed the impact on rotavirus factories. We found that the change caused the formation of smaller and more numerous factories that could not readily fuse together like those of the wild-type virus. The altered NSP2 protein also had a reduced capacity to form factory-like condensates in a test tube. Together, these results add to our growing understanding of how NSP2 supports rotavirus factory formation-a key step of viral replication.

Trivalent SARS-CoV-2 S1 Subunit Protein Vaccination Induces Broad Humoral Responses in BALB/c Mice.

This paper presents a novel approach for improving the efficacy of COVID-19 vaccines against emergent SARS-CoV-2 variants. We have evaluated the immunogenicity of unadjuvanted wild-type (WU S1-RS09cg) and variant-specific (Delta S1-RS09cg and OM S1-RS09cg) S1 subunit protein vaccines delivered either as a monovalent or a trivalent antigen in BALB/c mice. Our results show that a trivalent approach induced a broader humoral response with more coverage against antigenically distinct variants, especially when compared to monovalent Omicron-specific S1. This trivalent approach was also found to have increased or equivalent ACE2 binding inhibition, and increased S1 IgG endpoint titer at early timepoints, against SARS-CoV-2 spike variants when compared monovalent Wuhan, Delta, or Omicron S1. Our results demonstrate the utility of protein subunit vaccines against COVID-19 and provide insights into the impact of variant-specific COVID-19 vaccine approaches on the immune response in the current SARS-CoV-2 variant landscape. Particularly, our study provides insight into effects of further increasing valency of currently approved SARS-CoV-2 vaccines, a promising approach for improving protection to curtail emerging viral variants.

Monoclonal antibodies constructed from COVID-19 convalescent memory B cells exhibit potent binding activity to MERS-CoV spike S2 subunit and other human coronaviruses.

The Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are two highly contagious coronaviruses causing MERS and COVID-19, respectively, without an effective antiviral drug and a long-lasting vaccine. Approaches for diagnosis, therapeutics, prevention, etc., particularly for SARS-CoV-2 that is continually spreading and evolving, are urgently needed. Our previous study discovered that >60% of sera from convalescent COVID-19 individuals, but <8% from general population, showed binding activity against the MERS-CoV spike protein, indicating that SARS-CoV-2 infection boosted antibodies cross-reactive with MERS-CoV. To generate antibodies specific to both SARS-CoV-2 and MERS-CoV, here we screened 60 COVID-19 convalescent sera against MERS-CoV spike extracellular domain and S1 and S2 subunits. We constructed and characterized monoclonal antibodies (mAbs) from COVID-19 convalescent memory B cells and examined their binding and neutralizing activities against human coronaviruses. Of 60 convalescent serum samples, 34 showed binding activity against MERS-CoV S2, with endpoint titers positively correlated with the titers to SARS-CoV-2 S2. By sorting single memory B cells from COVID-19 convalescents, we constructed 38 mAbs and found that 11 mAbs showed binding activity with MERS-CoV S2, of which 9 mAbs showed potent cross-reactivity with all or a proportion of spike proteins of alphacoronaviruses (229E and NL63) and betacoronaviruses (SARS-CoV-1, SARS-CoV-2, OC43, and HKU1). Moreover, 5 mAbs also showed weak neutralization efficiency against MERS-CoV spike pseudovirus. Epitope analysis revealed that 3 and 8 mAbs bound to linear and conformational epitopes in MERS-CoV S2, respectively. In summary, we have constructed a panel of antibodies with broad-spectrum reactivity against all seven human coronaviruses, thus facilitating the development of diagnosis methods and vaccine design for multiple coronaviruses.

Immunogenicity and antigenicity of a conserved fragment of the rhoptry-associated membrane antigen of Plasmodium vivax

BackgroundPlasmodium vivax rhoptry-associated membrane antigen (RAMA) is a glycophosphatidylinositol-anchored membrane protein currently under consideration as a malaria vaccine candidate. Immunoglobulin G (IgG) antibodies induced by P. vivax RAMA (PvRAMA) have been proved to persist over 12 months in the sera of people infected with P. vivax. It has also been shown that through stimulation of peripheral blood mononuclear cells with PvRAMA in vitro, the antigen can induce CD4+ T cells to produce interleukin-10. However, the genetic diversity of the RAMA gene in isolates of P. vivax (pvrama) and the immunogenicity of PvRAMA in animals remain unclear.MethodsGenomic DNA was extracted from blood samples (n = 25) of patients in Jiangsu Province, China with imported infections of P. vivax from endemic countries in South and Southeast Asia. The extract genomic DNA was used as templates to amplify the P. vivax rama gene (pvrama) by PCR, and the PCR products were then sequenced and analyzed by the DnaSP, MEGA, and GeneDoc software packages. Recombinant PvRAMA (rPvRAMA) protein was expressed and purified, and then used to immunize mice. Levels of total IgG and different IgG subclasses of rPvRAMA-immunized mice were evaluated by enzyme-linked immunosorbent assay. Also, spleen cells of rPvRAMA-immunized mice were stimulated with rPvRAMA in vitro and levels of T cells were measured by flow cytometry.ResultsThe average pairwise nucleotide diversity (π) of the pvrama gene was 0.00190, and the haplotype diversity (Hd) was 0.982. The C-terminal of PvRAMA showed lower haplotype diversity compared to the N-terminal and was completely conserved at amino acid sites related to erythrocyte binding. To further characterize immunogenicity of the C-terminal of PvRAMA, mice were immunized with rPvRAMA antigen. The rPvRAMA protein induced antibody responses, with the end-point titer ranging from 1:10,000 to 1:5,120,000. IgG1 was the predominant IgG subclass in rPvRAMA-immunized mice, followed by IgG2b. In addition, levels of CD4+ and CD8+ T cells in the rPvRAMA-stimulated group were significantly higher than those in the phosphate-buffered saline-stimulated group (normal control group).ConclusionsThe high conservation at specific amino acid sites and the high immunogenicity of the C-terminal of PvRAMA indicate the presence of conserved epitopes able to generate broadly reactive humoral and cellular immune responses. These findings support the potential of PvRAMA to serve as a vaccine candidate against P. vivax infection.Graphical

Serosurvey of spotted fever group Rickettsia in equids from western Pará, Amazon, Brazil.

From a previous large epidemiological survey, we randomly selected 474 serum samples (463 horses and 11 mules) distributed among four municipalities of Pará state, Amazon region, Brazil, and from three types: farm animal, urban carthorse, and sport horse. Samples were tested by indirect fluorescence antibody test (IFAT ≥ 64) for antibodies reactive to spotted fever group (SFG) rickettsiae using Rickettsia rickettsii as crude antigens. From the 474 equids tested, 149 (31.4%) had ticks attached during sampling, belonging mostly to the species Dermacentor nitens. The overall seroprevalence for SFG rickettsiae was 31.4% (95% confidence interval: 27.3-35.9%) with 149 seropositive animals out of 474 screened. Notably, 77 equids (16.2%) had high endpoint titers ranging from 512 to 16,384, indicating that they had been exposed to SFG rickettsiae not long before sampling. Animal type affected rickettsial seroprevalence, with significantly higher values among farm horses when compared with urban and sport animals. Presence of dogs and tick infestation were negatively associated with equid seropositivity to R. rickettsii. This is the first report of SFG rickettsiae-reactive antibodies in equids from Pará state, Brazilian Amazon.

Inactivated genotype 1a, 2a and 3a HCV vaccine candidates induced broadly neutralising antibodies in mice

ObjectiveA prophylactic vaccine is needed to control the HCV epidemic, with genotypes 1–3 causing >80% of worldwide infections. Vaccine development is hampered by HCV heterogeneity, viral escape including protection of...

Cloning, expression and purification of Plasmodium knowlesi circumsporozoite protein and immunoblot analysis with P. knowlesi strain A1H1 protein extract.

Circumsporozoite protein (CSP) is a sporozoite major surface protein of Plasmodium species. The protein showed promising protection level as a vaccine candidate against Plasmodium falciparum infection. There is a lack of studies on P. knowlesi CSP (PkCSP) as a vaccine candidate due to the high polymorphic characteristic of central repeat region. Recent studies showed the protein has a relatively conserved region at the C-terminal, which consists of T- and B-cell epitopes. This could be the target region for vaccine development against the pre-erythrocytic stage of the parasite. In this study, recombinant PkCSP was expressed using Escherichia coli system. Recombinant PkCSP was immunized in animal models and the antiserum was evaluated using immunoblot analysis. Results showed that PkCSP can be successfully expressed using the bacterial system. Endpoint titre of the antiserum were ranged up to 1:819200. Immunoblot analysis showed the antiserum recognized recombinant PkCSP but not total protein extract from P. knowlesi erythrocytic stage. In conclusion, PkCSP could elicit strong immune response in animal models. However, serum antibodies could not recognize protein from the parasite's erythrocytic stage extract indicating it is not expressed at the erythrocytic stage. Therefore, PkCSP remains as a potential pre-erythrocytic vaccine candidate against P. knowlesi infection.

Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes.

ABSTRACTSpecies A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously.IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

Comparative Profiles of SARS-CoV-2 Spike-Specific Human Milk Antibodies Elicited by mRNA- and Adenovirus-Based COVID-19 Vaccines.

Background: Numerous COVID-19 vaccines are authorized globally. To date, ∼71% of doses comprise the Pfizer/BioNTech vaccine, and ∼17% the Moderna/NIH vaccine, both of which are messenger RNA (mRNA) based. The chimpanzee Ad-based Oxford/AstraZeneca (AZ) vaccine comprises ∼9%, while the Johnson & Johnson/Janssen (J&J) human adenovirus (Ad26) vaccine ranks fourth at ∼2%. No COVID-19 vaccine is yet available for children 0-4. One method to protect this population may be passive immunization through antibodies (Abs) provided in the milk of a lactating vaccinated person. Our early work and other reports have demonstrated that unlike the post-SARS-CoV-2 infection milk Ab profile, which is rich in specific secretory (s)IgA, the vaccine response is highly IgG dominant. Results: In this report, we present a comparative assessment of the milk Ab response elicited by Pfizer, Moderna, J&J, and AZ vaccines. This analysis revealed 86-100% of mRNA vaccine recipient milk exhibited Spike-specific IgG endpoint titers, which were 12- to 28-fold higher than those measured for Ad vaccine recipient milk. Ad-based vaccines elicited Spike-specific milk IgG in only 33-38% of recipients. Specific IgA was measured in 52-71% of mRNA vaccine recipient milk and 17-23% of Ad vaccine recipient milk. J&J recipient milk exhibited significantly lower IgA than Moderna recipients, and AZ recipients exhibited significantly lower IgA titers than Moderna and Pfizer. Less than 50% of milk of any group exhibited specific secretory Ab, with Moderna recipient IgA titers measuring significantly higher than AZ. Moderna appeared to most frequently elicit greater than twofold increases in specific secretory Ab titer relative to prevaccine sample. Conclusion: These data indicate that current Ad-based COVID-19 vaccines poorly elicit Spike-specific Ab in milk compared to mRNA-based vaccines, and that mRNA vaccines are preferred for immunizing the lactating population. This study highlights the need to design vaccines better aimed at eliciting an optimal milk Ab response.

Age and gender associated changes in immunoglobulin subclass levels specific to S. pneumoniae, serotype 1

S. pneumoniae is an important human pathogen which has a polysaccharide capsule with virulent properties. This work aims to estimate the titres of S. pneumoniae specific IgG and IgA isotypes, with respect to age and sex.An in-house whole bacterial cell ELISA was used for the determination of relative levels and endpoint titres of IgG subclasses and IgA1 subclass specific for S. pneumoniae serogroup 1, and to quantify specific IgG1 and IgG2 levels.Significantly lower anti-pneumococcus IgG1 titres were found in older individuals, which was more pronounced in men. Lower IgG2 titres were detected in men over 50 years of age, in comparison to women under 50 years of age. The levels of IgG3 and IgG4 did not differ between different sex and age groups. Lower IgA1 levels were detected in male individuals in both age groups in comparison to females under 50 years of age. The levels of IgG1 showed a moderate correlation with IgG4 in younger individuals of both sexes (r = 0.61 in men and 0.63 in women) which was not noted in the older age group.We highlight the deficiency in humoral immunity in older people, especially male and suggest immunization of this population with pneumococcal vaccines.

Phage-like particle vaccines are highly immunogenic and protect against pathogenic coronavirus infection and disease

The response by vaccine developers to the COVID-19 pandemic has been extraordinary with effective vaccines authorized for emergency use in the United States within 1 year of the appearance of the first COVID-19 cases. However, the emergence of SARS-CoV-2 variants and obstacles with the global rollout of new vaccines highlight the need for platforms that are amenable to rapid tuning and stable formulation to facilitate the logistics of vaccine delivery worldwide. We developed a “designer nanoparticle” platform using phage-like particles (PLPs) derived from bacteriophage lambda for a multivalent display of antigens in rigorously defined ratios. Here, we engineered PLPs that display the receptor-binding domain (RBD) protein from SARS-CoV-2 and MERS-CoV, alone (RBDSARS-PLPs and RBDMERS-PLPs) and in combination (hCoV-RBD PLPs). Functionalized particles possess physiochemical properties compatible with pharmaceutical standards and retain antigenicity. Following primary immunization, BALB/c mice immunized with RBDSARS- or RBDMERS-PLPs display serum RBD-specific IgG endpoint and live virus neutralization titers that, in the case of SARS-CoV-2, were comparable to those detected in convalescent plasma from infected patients. Further, these antibody levels remain elevated up to 6 months post-prime. In dose-response studies, immunization with as little as one microgram of RBDSARS-PLPs elicited robust neutralizing antibody responses. Finally, animals immunized with RBDSARS-PLPs, RBDMERS-PLPs, and hCoV-RBD PLPs were protected against SARS-CoV-2 and/or MERS-CoV lung infection and disease. Collectively, these data suggest that the designer PLP system provides a platform for facile and rapid generation of single and multi-target vaccines.

Immunogenicity Analysis of the Recombinant Plasmodium falciparum Surface-Related Antigen in Mice.

Plasmodium falciparum, mainly distributed in tropical and subtropical regions of the world, has received widespread attention owing to its severity. As a novel protein, P. falciparum surface-related antigen (PfSRA) has the structural and functional characteristics to be considered as a malaria vaccine candidate; however, limited information is available on its immunogenicity. Here, we expressed three fragments of recombinant PfSRA in an Escherichia coli system and further analyzed its immunogenicity. The results showed that rPfSRA-immunized mice produced specific antibodies with high endpoint titers (1:10,000 to 1:5,120,000) and affinity antibodies (i.e., rPfSRA-F1a (97.70%), rPfSRA-F2a (69.62%), and rPfSRA-F3a (91.87%)). In addition, the sera of immunized mice recognized both the native PfSRA and recombinant PfSRA, the rPfSRA antibodies inhibited the invasion of P. falciparum into the erythrocytes, and they were dose-dependent in vitro. This study confirmed PfSRA could be immunogenic, especially the F1a at the conserved region N-terminal and provided further support for it as a vaccine candidate against P. falciparum.

Safety and immunogenicity of inactivated SARS-CoV-2 vaccines in people living with HIV

ABSTRACT It is important to know the safety and efficacy of vaccination in immunocompromised people living with HIV (PLWH), but currently, there is limited data on the inactivated SARS-CoV-2 vaccines’ safety and immune responses in PLWH. In this prospective observational study, 139 PLWH and 120 healthy controls were enrolled and monitored for 21–105 days after a two-dose vaccination. The safety, anti-receptor binding domain IgG (anti-RBD-IgG) and anti-spike-IgG responses, and RBD-specific memory B cell (MBC) responses were evaluated. The overall adverse events within seven days were reported in 12.9% (18/139) of PLWH and 13.3% (16/120) of healthy controls. No serious adverse events occurred in both groups. Overall, the seroprevalence of anti-RBD-IgG in PLWH was significantly decreased (87.1% vs. 99.2%; p<0.001). The geometric mean end-point titer (GMT) of anti-RBD-IgG in PLWH was also reduced, especially in patients with CD4 counts <200 cells/µL, regardless of age, gender, or HIV viral load. GMTs of anti-RBD-IgG in both PLWH and healthy controls declined gradually over time. Similar results were also observed in the anti-spike-IgG response. The frequency of RBD-specific MBCs in PLWH decreased (p<0.05), and then remained stable over time. Lastly, through multivariate analysis, we found the factors that predicted a less robust response to inactivated vaccines in PLWH were a low CD4 count and long time interval after vaccination. In conclusion, inactivated vaccines are well-tolerated in PLWH but with low immunogenicity. Therefore, SARS-CoV-2 vaccines and booster doses should be given priority in PLWH, especially in patients with low CD4 counts. Trial registration: ClinicalTrials.gov identifier: NCT05043129..

Affordable mobile microfluidic diagnostics: minimum requirements for smartphones and digital imaging for colorimetric and fluorometric anti-dengue and anti-SARS-CoV-2 antibody detection

Background: Miniaturised bioassays permit diagnostic testing near the patient, and the results can be recorded digitally using inexpensive cameras including smartphone and mobile phone cameras. Although digital cameras are now inexpensive and portable, the minimum performance required for microfluidic diagnostic bioassays has not been defined. We present a systematic comparison of a wide range of different digital cameras for capturing and measuring results of microfluidic bioassays and describe a framework to specify performance requirements to quantify immunoassays. Methods: A set of 200 µm diameter microchannels was filled with a range of concentrations of dyes used in colorimetric and fluorometric enzyme immunoassays. These were imaged in parallel using cameras of varying cost and performance ranging from <£30 to >£500. Results: Higher resolution imaging allowed larger numbers of microdevices to be resolved and analysed in a single image. In contrast, low quality cameras were still able to quantify results but for fewer samples. In some cases, an additional macro lens was added to focus closely. If image resolution was sufficient to identify individual microfluidic channels as separate lines, all cameras were able to quantify a similar range of concentrations of both colorimetric and fluorometric dyes. However, the mid-range cameras performed better, with the lowest cost cameras only allowing one or two samples to be quantified per image. Consistent with these findings, we demonstrate that quantitation (to determine endpoint titre) of antibodies against dengue and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses is possible using a wide range of digital imaging devices including the mid-range smartphone iPhone 6S and a budget Android smartphone costing <£50. Conclusions: In conclusion, while more expensive and higher quality cameras allow larger numbers of devices to be simultaneously imaged, even the lowest resolution and cheapest cameras were sufficient to record and quantify immunoassay results.