Making sense of emerging evidence on the non-specific effects of the BCG vaccine on malaria risk and neonatal mortality

Vaccines are, indisputably, one of the greatest public health interventions, with a substantial positive impact on child survival. The remarkable declines in child mortality observed during the last quarter of a century, whereby global under 5 deaths were essentially halved, go hand in hand with the estimated 2–3 million child deaths prevented by vaccines annually.1 The premise for this is clear: vaccines directly prevent a variety of life-threatening diseases. Vaccines can also be held directly responsible for the eradication of smallpox, the first and only infectious disease extinguished by the action of humans and are paving the way for the disappearance of other terrible infections such as polio, measles or rubella

Development of quantitative suspension array assays for six immunoglobulin isotypes and subclasses to multiple Plasmodium falciparum antigens

Background Quantitative suspension arrays are powerful immunoassays to measure antibodies against multiple antigens in large numbers of samples in a short time and using few microliters. To identify antigen targets of immunity for vaccine development against complex microbes like Plasmodium falciparum, such technology allows the characterization of the magnitude and antigenic specificity of Ig isotypes and subclasses that are important for functional responses. However, standardized assays are not widely available. Methods We developed six quantitative suspension array assays to measure IgG1, IgG2, IgG3, IgG4, IgM and IgE specific to multiple P. falciparum antigens. Secondary and tertiary antibodies, as well as human purified antibodies for standard curves, were tested among several commercially available sources. Positive and negative controls included plasmas from malaria hyper-immune African adults and from malaria-naïve European adults, respectively. Reagents were selected and optimal antibody and test sample dilutions established according to sensitivity, specificity and performance of the standard curves. The variability between replicates and plates was assessed with 30 test samples and controls. Results Assays were able to detect P. falciparum antigen-specific antibodies for all isotypes and subclasses in samples from malaria-exposed individuals, with low background signal in blank wells. Levels detected in malaria-naïve individuals were overall low except for IgM. For the IgG2 and IgE assays, a triple sandwich was required for sensitivity. Standard curves with 5-parameter logistic fit were successfully obtained in all assays. The coefficients of variation for measurements performed in different days were all <30%, and <5% when comparing duplicates from the same plate. Conclusion The isotype/subclass assays developed here were sensitive, specific, reproducible and of adequate quantification dynamic range. They allow performing detailed immuno-profiling to large panels of P. falciparum antigens to address naturally- and vaccine-induced Ig responses and elucidate correlates of malaria protection, and could also be applied to other antigenic panels

Comparison of commercial kits to measure cytokine responses to Plasmodium falciparum by multiplex microsphere suspension array technology

<p>Abstract</p> <p>Background</p> <p>Multiplex cytokine profiling systems are useful tools for investigating correlates of protective immunity. Several Luminex and flow cytometry methods are commercially available but there is limited information on the relative performance of different kits. A series of comparison experiments were carried out to determine the most appropriate method for our subsequent studies.</p> <p>Methods</p> <p>Two Luminex methods were compared, the Bio-Rad human 17-plex panel and the Invitrogen (formerly BioSource) human cytokine 10-plex kit, and two flow cytometry methods, the Becton Dickinson Human Th1/Th2 Cytokine Kit (CBA) and the Bender MedSystems Human Th1/Th2 11plex FlowCytomix Multiplex Kit. All kits were tested for the measurement of cytokines in supernatants collected from human leukocytes stimulated with viable <it>Plasmodium falciparum </it>infected red blood cells (iRBC) or <it>P. falciparum </it>schizont lysates.</p> <p>Results</p> <p>Data indicated that the kits differed in sensitivity and reproducibility depending on the cytokine, and detected different quantities of some cytokines. The Bio-Rad 17-plex kit was able to detect more positive responses than the Invitrogen 10-plex kit. However, only when detecting IL-1, IL-6 or TNF did the two Luminex based methods correlate with one another. In this study, the flow cytometry based techniques were less variable and correlated better with one another. The two flow cytometry based kits showed significant correlation when detecting IFN-γ, IL-2, TNF, IL-10 and IL-6, but overall the BD kit detected more positive responses than the Bender MedSystems kit.</p> <p>Conclusions</p> <p>The microsphere suspension array technologies tested differed in reproducibility and the absolute quantity of cytokine detected. Sample volume, the number of cytokines measured, and the time and cost of the assays also differed. These data provide an accurate assessment of the four techniques, which will allow individual researchers to select the tool most suited for their study population.</p

Immunosuppressive and angiogenic cytokine profile associated with Bartonella bacilliformis infection in post-outbreak and endemic areas of Carrion's disease in Peru

Analysis of immune responses in Bartonella bacilliformis carriers are needed to understand acquisition of immunity to Carrion's disease and may allow identifying biomarkers associated with bacterial infection and disease phases. Serum samples from 144 healthy subjects from 5 villages in the North of Peru collected in 2014 were analyzed. Four villages had a Carrion's disease outbreak in 2013, and the other is a traditionally endemic area. Thirty cytokines, chemokines and growth factors were determined in sera by fluorescent bead-based quantitative suspension array technology, and analyzed in relation to available data on bacteremia quantified by RT-PCR, and IgM and IgG levels measured by ELISA against B. bacilliformis lysates. The presence of bacteremia was associated with low concentrations of HGF (p = 0.005), IL-15 (p = 0.002), IL-6 (p = 0.05), IP-10 (p = 0.008), MIG (p = 0.03) and MIP-1alpha (p = 0.03). In multi-marker analysis, the same and further TH1-related and pro-inflammatory biomarkers were inversely associated with infection, whereas angiogenic chemokines and IL-10 were positively associated. Only EGF and eotaxin showed a moderate positive correlation with bacteremia. IgM seropositivity, which reflects a recent acute infection, was associated with lower levels of eotaxin (p = 0.05), IL-6 (p = 0.001), and VEGF (p = 0.03). Only GM-CSF and IL-10 concentrations were positively associated with higher levels of IgM (p = 0.01 and p = 0.007). Additionally, IgG seropositivity and levels were associated with high levels of angiogenic markers VEGF (p = 0.047) and eotaxin (p = 0.006), respectively. Our findings suggest that B. bacilliformis infection causes immunosuppression, led in part by overproduction of IL-10. This immunosuppression probably contributes to the chronicity of asymptomatic infections favoring B. bacilliformis persistence in the host, allowing the subsequent transmission to the vector. In addition, angiogenic markers associated with bacteremia and IgG levels may be related to the induction of endothelial cell proliferation in cutaneous lesions during chronic infections, being possible candidate biomarkers of asymptomatic infections

Improving Vaccine-Induced Immunity: Can Baseline Predict Outcome?

Immune signatures measured at baseline and immediately prior to vaccination may predict the immune response to vaccination. Such pre-vaccine assessment might allow not only population-based, but also more personalized vaccination strategies ('precision vaccination'). If baseline immune signatures are predictive, the underlying mechanism they reflect may also determine vaccination outcome. Thus, baseline signatures might contribute to identifying interventional targets to be modulated prior to vaccination in order to improve vaccination responses. This concept has the potential to transform vaccination strategies and usher in a new approach to improve global health

VAR2CSA Signatures of High Plasmodium falciparum Parasitemia in the Placenta

Plasmodium falciparum infected erythrocytes (IE) accumulate in the placenta through the interaction between Duffy-binding like (DBL) domains of parasite-encoded ligand VAR2CSA and chondroitin sulphate-A (CSA) receptor. Polymorphisms in these domains, including DBL2X and DBL3X, may affect their antigenicity or CSA-binding affinity, eventually increasing parasitemia and its adverse effects on pregnancy outcomes. A total of 373 DBL2X and 328 DBL3X sequences were obtained from transcripts of 20 placental isolates infecting Mozambican women, resulting in 176 DBL2X and 191 DBL3X unique sequences at the protein level. Sequence alignments were divided in segments containing combinations of correlated polymorphisms and the association of segment sequences with placental parasite density was tested using Bonferroni corrected regression models, taking into consideration the weight of each sequence in the infection. Three DBL2X and three DBL3X segments contained signatures of high parasite density (P<0.003) that were highly prevalent in the parasite population (49–91%). Identified regions included a flexible loop that contributes to DBL3X-CSA interaction and two DBL3X motifs with evidence of positive natural selection. Limited antibody responses against signatures of high parasite density among malaria-exposed pregnant women could not explain the increased placental parasitemia. These results suggest that a higher binding efficiency to CSA rather than reduced antigenicity might provide a biological advantage to parasites with high parasite density signatures in VAR2CSA. Sequences contributing to high parasitemia may be critical for the functional characterization of VAR2CSA and the development of tools against placental malaria

Induction and decay of functional complement-fixing antibodies by the RTS,S malaria vaccine in children, and a negative impact of malaria exposure

Background: Leading malaria vaccine, RTS,S, is based on the circumsporozoite protein (CSP) of sporozoites. RTS,S confers partial protection against malaria in children, but efficacy wanes relatively quickly after primary immunization. Vaccine efficacy has some association with anti-CSP IgG; however, it is unclear how these antibodies function, and how functional antibodies are induced and maintained over time. Recent studies identified antibodycomplement interactions as a potentially important immune mechanism against sporozoites. Here, we investigated whether RTS,S vaccine-induced antibodies could function by interacting with complement. Methods: Serum samples were selected from children in a phase IIb trial of RTS,S/AS02A conducted at two study sites of high and low malaria transmission intensity in Manhiça, Mozambique. Samples following primary immunization and 5-year post-immunization follow-up time points were included. Vaccine-induced antibodies were characterized by isotype, subclass, and epitope specificity, and tested for the ability to fix and activate complement. We additionally developed statistical methods to model the decay and determinants of functional antibodies after vaccination. Results: RTS,S vaccination induced anti-CSP antibodies that were mostly IgG1, with some IgG3, IgG2, and IgM. Complement-fixing antibodies were effectively induced by vaccination, and targeted the central repeat and Cterminal regions of CSP. Higher levels of complement-fixing antibodies were associated with IgG that equally recognized both the central repeat and C-terminal regions of CSP. Older age and higher malaria exposure were significantly associated with a poorer induction of functional antibodies. There was a marked decay in functional complement-fixing antibodies within months after vaccination, as well as decays in IgG subclasses and IgM. Statistical modeling suggested the decay in complement-fixing antibodies was mostly attributed to the waning of anti-CSP IgG1, and to a lesser extent IgG3. Conclusions: We demonstrate for the first time that RTS,S can induce complement-fixing antibodies in young malaria-exposed children. The short-lived nature of functional responses mirrors the declining vaccine efficacy of RTS,S over time. The negative influence of age and malaria exposure on functional antibodies has implications for understanding vaccine efficacy in different settings. These findings provide insights into the mechanisms and longevity of vaccine-induced immunity that will help inform the future development of highly efficacious and longlasting malaria vaccines

Decreased and Heterogeneous Neutralizing Antibody Responses Against RBD of SARS-CoV-2 Variants After mRNA Vaccination.

The rapid spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerging variants raises concerns about their capacity to evade immune protection provided by natural infection or vaccination. The receptor-binding domain (RBD) of the viral spike protein is the major target of neutralizing antibodies, and viral variants accumulate mutations in this region. In this study, we determined the antibody neutralization capacity against the RBD of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Epsilon (B.1.427), Kappa (B.1.617.1), and Delta (B.1.617.2) in a cohort of healthcare workers naturally infected or receiving COVID-19 mRNA vaccines from Moderna or Pfizer-BioNTech. We show that the five RBD variants displayed an augmented binding to ACE2 compared to the original Wuhan strain. The most significant increase was observed in variants Epsilon and Delta, containing mutation L452R. Using a flow cytometry cell-based assay, we found that SARS-CoV-2-infected subjects presented low levels of RBD-specific neutralizing antibodies against all variants analyzed, except Alpha. However, the neutralizing activity incremented considerably after a subsequent mRNA-vaccine dose, to levels significantly higher than those in naïve individuals receiving two vaccine doses. Importantly, we observed partially impaired neutralizing responses against most variants in fully vaccinated individuals. Variants Gamma and Kappa encompassing RBD E484K/Q mutations presented the highest neutralizing resistance. Furthermore, a wide heterogeneity in the magnitude of RBD-specific neutralizing responses against all tested SARS-CoV-2 variants following both mRNA vaccines was detected. Altogether, our findings provide important knowledge regarding SARS-CoV-2 vaccine-induced immunity, and should be very useful to guide future vaccination regimens and personalized vaccine approaches

Changing plasma cytokine, chemokine and growth factor profiles upon differing malaria transmission intensities

Background: Malaria epidemiological and immunological data suggest that parasite tolerance wanes in the absence of continuous exposure to the parasite, potentially enhancing pathogenesis. The expansion of control interventions and elimination campaigns raises the necessity to better understand the host factors leading to susceptibility or tolerance that are afected by rapid changes in malaria transmission intensity (MTI). Mediators of cellular immune responses are responsible for the symptoms and pathological alterations during disease and are expected to change rapidly upon malaria exposure or cessation. Methods: The plasma concentrations of 30 cytokine, chemokine and growth factors in individuals of all ages from a malaria endemic area of southern Mozambique were compared between 2 years of diferent MTI: 2010 (lower, n=234) and 2013 (higher, n=143). The efect of the year on the correlations between cytokines, chemokines and growth factors and IgGs to Plasmodium falciparum (markers of exposure) was explored. The efects of age, sex, neighbourhood and parasitaemia on analyte levels and their interactions with year were also assessed. Results: An inverse correlation of several cellular immune mediators with malarial antibodies in 2013, and a lack of correlation or even a positive correlation in 2010 were observed. Most cytokines, chemokines and growth factors, regardless of their immune function, had higher concentrations in 2010 compared with 2013 in P. falciparum-infected and uninfected subjects. Age and neighbourhood showed an efect on analyte concentrations. Conclusions: The results show a diferent regulation of the cellular immune response in 2010 vs 2013 which could be related to a loss of immune-tolerance after a decline in MTI in 2010 and previous years, and a rapid re-establishment of tolerance as a consequence of more continuous exposure as MTI began increasing in 2012. Cellular immune mediators warrant further investigation as possible surrogates of MTI-associated host susceptibility or tolerance

An Autopsy Study of Maternal Mortality in Mozambique: The Contribution of Infectious Diseases

Clara Menendez and colleagues analyze 139 complete autopsies following maternal deaths in Maputo, Mozambique and find a predominance of infectious and preventable causes