Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray.

The evaluation of protein antigens as putative serologic biomarkers of infection has increasingly shifted to high-throughput, multiplex approaches such as the protein microarray. In vitro transcription/translation (IVTT) systems-a similarly high-throughput protein expression method-are already widely utilised in the production of protein microarrays, though purified recombinant proteins derived from more traditional whole cell based expression systems also play an important role in biomarker characterisation. Here we have performed a side-by-side comparison of antigen-matched protein targets from an IVTT and purified recombinant system, on the same protein microarray. The magnitude and range of antibody responses to purified recombinants was found to be greater than that of IVTT proteins, and responses between targets from different expression systems did not clearly correlate. However, responses between amino acid sequence-matched targets from each expression system were more closely correlated. Despite the lack of a clear correlation between antigen-matched targets produced in each expression system, our data indicate that protein microarrays produced using either method can be used confidently, in a context dependent manner, though care should be taken when comparing data derived from contrasting approaches.

Persistence of SARS-CoV-2 Antibodies in Vaccinated Health Care Workers Analyzed by Coronavirus Antigen Microarray.

Recent studies provide conflicting evidence on the persistence of SARS-CoV-2 immunity induced by mRNA vaccines. Here, we aim to quantify the persistence of humoral immunity following vaccination using a coronavirus antigen microarray that includes 10 SARS-CoV-2 antigens. In a prospective longitudinal cohort of 240 healthcare workers, composite SARS-CoV-2 IgG antibody levels did not wane significantly over a 6-month study period. In the subset of the study population previously exposed to SARS-CoV-2 based on seropositivity for nucleocapsid antibodies, higher composite anti-spike IgG levels were measured before the vaccine but no significant difference from unexposed individuals was observed at 6 months. Age, vaccine type, or worker role did not significantly impact composite IgG levels, although non-significant trends towards lower antibody levels in older participants and higher antibody levels with Moderna vaccine were observed at 6 months. A small subset of our cohort were classified as having waning antibody titers at 6 months, and these individuals were less likely to work in patient care roles and more likely to have prior exposure to SARS-CoV-2.

Distinct SARS-CoV-2 antibody reactivity patterns elicited by natural infection and mRNA vaccination.

We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8476 finger stick blood specimens were collected before and after a vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing antigens from SARS-CoV-2, SARS, MERS, Common CoV, and Influenza. Twenty-six percent of specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive; out of 852 seropositive individuals 77 had symptoms and 9 sought medical care. The antibody response was predominantly against nucleocapsid (NP), full length, and S2 domain of spike. Anti-receptor binding domain (RBD) reactivity was low and not cross-reactive against SARS S1 or SARS RBD. A vaccination campaign at the University of California Irvine Medical Center (UCIMC) started on December, 2020 and 6724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% pre-vaccination to 79% post-vaccination in January, 93% in February, and 99% in March. mRNA vaccination induced higher antibody levels than natural exposure, especially against the RBD domain and cross-reactivity against SARS RBD and S1 was observed. Nucleocapsid protein antibodies can be used to distinguish vaccinees to classify pre-exposure to SARS-CoV-2 Previously infected individuals developed higher antibody titers to the vaccine than non pre-exposed individuals. Hospitalized patients in intensive care with severe disease reach significantly higher antibody levels than mild cases, but lower antibody levels compared to the vaccine. These results indicate that mRNA vaccination rapidly induces a much stronger and broader antibody response than SARS-CoV-2 infection.

Immunoprofiles associated with controlled human malaria infection and naturally acquired immunity identify a shared IgA pre-erythrocytic immunoproteome.

Knowledge of the Plasmodium falciparum antigens that comprise the human liver stage immunoproteome is important for pre-erythrocytic vaccine development, but, compared with the erythrocytic stage immunoproteome, more challenging to classify. Previous studies of P. falciparum antibody responses report IgG and rarely IgA responses. We assessed IgG and IgA antibody responses in adult sera collected during two controlled human malaria infection (CHMI) studies in malaria-naïve volunteers and in 1- to 6-year-old malaria-exposed Malian children on a 251 P. falciparum antigen protein microarray. IgG profiles in the two CHMI groups were equivalent and differed from Malian children. IgA profiles were robust in the CHMI groups and a subset of Malian children. We describe immunoproteome differences in naïve vs. exposed individuals and report pre-erythrocytic proteins recognized by the immune system. IgA responses detected in this study expand the list of pre-erythrocytic antigens for further characterization as potential vaccine candidates.

Novel insights from the Plasmodium falciparum sporozoite-specific proteome by probabilistic integration of 26 studies.

Plasmodium species, the causative agent of malaria, have a complex life cycle involving two hosts. The sporozoite life stage is characterized by an extended phase in the mosquito salivary glands followed by free movement and rapid invasion of hepatocytes in the human host. This transmission stage has been the subject of many transcriptomics and proteomics studies and is also targeted by the most advanced malaria vaccine. We applied Bayesian data integration to determine which proteins are not only present in sporozoites but are also specific to that stage. Transcriptomic and proteomic Plasmodium data sets from 26 studies were weighted for how representative they are for sporozoites, based on a carefully assembled gold standard for Plasmodium falciparum (Pf) proteins known to be present or absent during the sporozoite life stage. Of 5418 Pf genes for which expression data were available at the RNA level or at the protein level, 975 were identified as enriched in sporozoites and 90 specific to them. We show that Pf sporozoites are enriched for proteins involved in type II fatty acid synthesis in the apicoplast and GPI anchor synthesis, but otherwise appear metabolically relatively inactive in the salivary glands of mosquitos. Newly annotated hypothetical sporozoite-specific and sporozoite-enriched proteins highlight sporozoite-specific functions. They include PF3D7_0104100 that we identified to be homologous to the prominin family, which in human has been related to a quiescent state of cancer cells. We document high levels of genetic variability for sporozoite proteins, specifically for sporozoite-specific proteins that elicit antibodies in the human host. Nevertheless, we can identify nine relatively well-conserved sporozoite proteins that elicit antibodies and that together can serve as markers for previous exposure. Our understanding of sporozoite biology benefits from identifying key pathways that are enriched during this life stage. This work can guide studies of molecular mechanisms underlying sporozoite biology and potential well-conserved targets for marker and drug development.

Analysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Blood using a Coronavirus Antigen Microarray.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray.

The current practice for diagnosis of SARS-CoV-2 infection relies on PCR testing of nasopharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk. This testing strategy likely underestimates the true prevalence of infection, creating the need for serologic methods to detect infections missed by the limited testing to date. Here, we describe the development of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A preliminary study of human sera collected prior to the SARS-CoV-2 pandemic demonstrates overall high IgG reactivity to common human coronaviruses and low IgG reactivity to epidemic coronaviruses including SARS-CoV-2, with some cross-reactivity of conserved antigenic domains including S2 domain of spike protein and nucleocapsid protein. This array can be used to answer outstanding questions regarding SARS-CoV-2 infection, including whether baseline serology for other coronaviruses impacts disease course, how the antibody response to infection develops over time, and what antigens would be optimal for vaccine development.

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens. This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera. Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

Distinct Antibody Signatures Associated with Different Malaria Transmission Intensities in Zambia and Zimbabwe.

Antibodies to Plasmodium falciparum are specific biomarkers that can be used to monitor parasite exposure over broader time frames than microscopy, rapid diagnostic tests, or molecular assays. Consequently, seroprevalence surveys can assist with monitoring the impact of malaria control interventions, particularly in the final stages of elimination, when parasite incidence is low. The protein array format to measure antibodies to diverse P. falciparum antigens requires only small sample volumes and is high throughput, permitting the monitoring of malaria transmission on large spatial and temporal scales. We expanded the use of a protein microarray to assess malaria transmission in settings beyond those with a low malaria incidence. Antibody responses in children and adults were profiled, using a P. falciparum protein microarray, through community-based surveys in three areas in Zambia and Zimbabwe at different stages of malaria control and elimination. These three epidemiological settings had distinct serological profiles reflective of their malaria transmission histories. While there was little correlation between transmission intensity and antibody signals (magnitude or breadth) in adults, there was a clear correlation in children younger than 5 years of age. Antibodies in adults appeared to be durable even in the absence of significant recent transmission, whereas antibodies in children provided a more accurate picture of recent levels of transmission intensity. Seroprevalence studies in children could provide a valuable marker of progress toward malaria elimination.IMPORTANCE As malaria approaches elimination in many areas of the world, monitoring the effect of control measures becomes more important but challenging. Low-level infections may go undetected by conventional tests that depend on parasitemia, particularly in immune individuals, who typically show no symptoms of malaria. In contrast, antibodies persist after parasitemia and may provide a more accurate picture of recent exposure. Only a few parasite antigens-mainly vaccine candidates-have been evaluated in seroepidemiological studies. We examined antibody responses to 500 different malaria proteins in blood samples collected through community-based surveillance from areas with low, medium, and high malaria transmission intensities. The breadth of the antibody responses in adults was broad in all three settings and was a poor correlate of recent exposure. In contrast, children represented a better sentinel population for monitoring recent malaria transmission. These data will help inform the use of multiplex serology for malaria surveillance.

Antibody Biomarkers Associated with Sterile Protection Induced by Controlled Human Malaria Infection under Chloroquine Prophylaxis.

Immunization with sporozoites under chloroquine chemoprophylaxis (CPS) induces distinctly preerythrocytic and long-lasting sterile protection against homologous controlled human malaria infection (CHMI). To identify possible humoral immune correlates of protection, plasma samples were collected from 38 CPS-immunized Dutch volunteers for analysis using a whole Plasmodium falciparum proteome microarray with 7,455 full-length or segmented protein features displaying about 91% of the total P. falciparum proteome. We identified 548 reactive antigens representing 483 unique proteins. Using the breadth of antibody responses for each subject in a mixture-model algorithm, we observed a trimodal pattern, with distinct groups of 16 low responders, 19 medium responders, and 3 high responders. Fifteen out of 16 low responders, 12 of the 19 medium responders, and 3 out of 3 high responders were fully protected from a challenge infection. In the medium-responder group, we identified six novel antigens associated with protection (area under the curve [AUC] value of ≥0.75; P < 0.05) and six other antigens that were specifically increased in nonprotected volunteers (AUC value of ≤0.25; P < 0.05). When used in combination, the multiantigen classifier predicts CPS-induced protective efficacy with 83% sensitivity and 88% specificity. The antibody response patterns characterized in this study represent surrogate markers that may provide rational guidance for clinical vaccine development.IMPORTANCE Infection by Plasmodium parasites has been a major cause of mortality and morbidity in humans for thousands of years. Despite the considerable reduction of deaths, according to the WHO, over 5 billion people are still at risk, with about 216 million worldwide cases occurring in 2016. More compelling, 15 countries in sub-Saharan Africa bore 80% of the worldwide malaria burden. Complete eradication has been challenging, and the development of an affordable and effective vaccine will go a long way in achieving elimination. However, identifying vaccine candidate targets has been difficult. In the present study, we use a highly effective immunization protocol that confers long-lasting sterile immunity in combination with a whole P. falciparum proteome microarray to identify antibody responses associated with protection. This study characterizes a novel antibody profile associated with sterile protective immunity and trimodal humoral responses that sheds light on the possible mechanism of CPS-induced immunity against P. falciparum parasites.

Protein Microarray Analysis of the Specificity and Cross-Reactivity of Influenza Virus Hemagglutinin-Specific Antibodies.

Current seasonal influenza virus vaccines engender antibody-mediated protection that is hemagglutinin (HA) subtype specific and relatively short-lived. Coverage for other subtypes or even variants within a subtype could be improved from a better understanding of the factors that promote HA-specific antibody cross-reactivity. Current assays to evaluate cross-reactivity, such as the ELISA, require a separate test for each antigen and are neither high-throughput nor sample-sparing. To address this need, we produced an array of 283 purified HA proteins from influenza A virus subtypes H1 to H16 and H18 and influenza B virus. To evaluate performance, arrays were probed with sera from individuals before and after a booster dose of inactivated heterologous H5N1 vaccine and naturally infected cases at presentation and follow-up during the 2010 to 2011 influenza season, when H3N2 was prevalent. The response to the H5 vaccine boost was IgG only and confined to H5 variants. The response to natural H3N2 infection consisted of IgG and IgA and was reactive with all H3 variants displayed, as well as against other group 2 HA subtypes. In both groups, responses to HA1 proteins were subtype specific. In contrast, baseline signals were higher, and responses broader, against full-length HA proteins (HA1+HA2) compared to HA1 alone. We propose that these elevated baseline signals and breadth come from the recognition of conserved epitopes in the stalk domain by cross-reactive antibodies accumulated from previous exposure(s) to seasonal influenza virus. This array is a valuable high-throughput alternative to the ELISA for monitoring specificity and cross-reactivity of HA antibodies and has many applications in vaccine development.IMPORTANCE Seasonal influenza is a serious public health problem because the viral infection spreads easily from person to person and because of antigenic drift in neutralizing epitopes. Influenza vaccination is the most effective way to prevent the disease, although challenging because of the constant evolution of influenza virus subtypes. Our high-throughput protein microarrays allow for interrogation of subunit-specific IgG and IgA responses to 283 different HA proteins comprised of HA1 and HA2 domains as well as full-length HA proteins. This provides a tool that allows for novel insights into the response to exposure to influenza virus antigens. Data generated with our technology will enhance our understanding of the factors that improve the strength, breadth, and durability of vaccine-mediated immune responses and develop more effective vaccines.

Safety, Immunogenicity, and Protective Efficacy of Intradermal Immunization with Aseptic, Purified, Cryopreserved Plasmodium falciparum Sporozoites in Volunteers Under Chloroquine Prophylaxis: A Randomized Controlled Trial.

Immunization of volunteers under chloroquine prophylaxis by bites of Plasmodium falciparum sporozoite (PfSPZ)-infected mosquitoes induces > 90% protection against controlled human malaria infection (CHMI). We studied intradermal immunization with cryopreserved, infectious PfSPZ in volunteers taking chloroquine (PfSPZ chemoprophylaxis vaccine [CVac]). Vaccine groups 1 and 3 received 3× monthly immunizations with 7.5 × 10(4) PfSPZ. Control groups 2 and 4 received normal saline. Groups 1 and 2 underwent CHMI (#1) by mosquito bite 60 days after the third immunization. Groups 3 and 4 were boosted 168 days after the third immunization and underwent CHMI (#2) 137 days later. Vaccinees (11/20, 55%) and controls (6/10, 60%) had the same percentage of mild to moderate solicited adverse events. After CHMI #1, 8/10 vaccinees (group 1) and 5/5 controls (group 2) became parasitemic by microscopy; the two negatives were positive by quantitative real-time polymerase chain reaction (qPCR). After CHMI #2, all vaccinees in group 3 and controls in group 4 were parasitemic by qPCR. Vaccinees showed weak antibody and no detectable cellular immune responses. Intradermal immunization with up to 3 × 10(5) PfSPZ-CVac was safe, but induced only minimal immune responses and no sterile protection against Pf CHMI.

Early effector cells survive the contraction phase in malaria infection and generate both central and effector memory T cells.

CD4 T cells orchestrate immunity against blood-stage malaria. However, a major challenge in designing vaccines to the disease is poor understanding of the requirements for the generation of protective memory T cells (Tmem) from responding effector T cells (Teff) in chronic parasite infection. In this study, we use a transgenic mouse model with T cells specific for the merozoite surface protein (MSP)-1 of Plasmodium chabaudi to show that activated T cells generate three distinct Teff subsets with progressive activation phenotypes. The earliest observed Teff subsets (CD127(-)CD62L(hi)CD27(+)) are less divided than CD62L(lo) Teff and express memory genes. Intermediate (CD62L(lo)CD27(+)) effector subsets include the most multicytokine-producing T cells, whereas fully activated (CD62L(lo)CD27(-)) late effector cells have a terminal Teff phenotype (PD-1(+), Fas(hi), AnnexinV(+)). We show that although IL-2 promotes expansion, it actually slows terminal effector differentiation. Using adoptive transfer, we show that only early Teff survive the contraction phase and generate the terminal late Teff subsets, whereas in uninfected recipients, they become both central and effector Tmem. Furthermore, we show that progression toward full Teff activation is promoted by increased duration of infection, which in the long-term promotes Tem differentiation. Therefore, we have defined markers of progressive activation of CD4 Teff at the peak of malaria infection, including a subset that survives the contraction phase to make Tmem, and show that Ag and cytokine levels during CD4 T cell expansion influence the proportion of activated cells that can survive contraction and generate memory in malaria infection.

Impact of malaria preexposure on antiparasite cellular and humoral immune responses after controlled human malaria infection.

To understand the effect of previous malaria exposure on antiparasite immune responses is important for developing successful immunization strategies. Controlled human malaria infections (CHMIs) using cryopreserved Plasmodium falciparum sporozoites provide a unique opportunity to study differences in acquisition or recall of antimalaria immune responses in individuals from different transmission settings and genetic backgrounds. In this study, we compared antiparasite humoral and cellular immune responses in two cohorts of malaria-naive Dutch volunteers and Tanzanians from an area of low malarial endemicity, who were subjected to the identical CHMI protocol by intradermal injection of P. falciparum sporozoites. Samples from both trials were analyzed in parallel in a single center to ensure direct comparability of immunological outcomes. Within the Tanzanian cohort, we distinguished one group with moderate levels of preexisting antibodies to asexual P. falciparum lysate and another that, based on P. falciparum serology, resembled the malaria-naive Dutch cohort. Positive P. falciparum serology at baseline was associated with a lower parasite density at first detection by quantitative PCR (qPCR) after CHMI than that for Tanzanian volunteers with negative serology. Post-CHMI, both Tanzanian groups showed a stronger increase in anti-P. falciparum antibody titers than Dutch volunteers, indicating similar levels of B-cell memory independent of serology. In contrast to the Dutch, Tanzanians failed to increase P. falciparum-specific in vitro recall gamma interferon (IFN-γ) production after CHMI, and innate IFN-γ responses were lower in P. falciparum lysate-seropositive individuals than in seronegative individuals. In conclusion, positive P. falciparum lysate serology can be used to identify individuals with better parasite control but weaker IFN-γ responses in circulating lymphocytes, which may help to stratify volunteers in future CHMI trials in areas where malaria is endemic.

Sporozoite immunization of human volunteers under mefloquine prophylaxis is safe, immunogenic and protective: a double-blind randomized controlled clinical trial.

Immunization of healthy volunteers with chloroquine ChemoProphylaxis and Sporozoites (CPS-CQ) efficiently and reproducibly induces dose-dependent and long-lasting protection against homologous Plasmodium falciparum challenge. Here, we studied whether chloroquine can be replaced by mefloquine, which is the only other licensed anti-malarial chemoprophylactic drug that does not affect pre-erythrocytic stages, exposure to which is considered essential for induction of protection by CPS immunization. In a double blind randomized controlled clinical trial, volunteers under either chloroquine prophylaxis (CPS-CQ, n = 5) or mefloquine prophylaxis (CPS-MQ, n = 10) received three sub-optimal CPS immunizations by bites from eight P. falciparum infected mosquitoes each, at monthly intervals. Four control volunteers received mefloquine prophylaxis and bites from uninfected mosquitoes. CPS-MQ immunization is safe and equally potent compared to CPS-CQ inducing protection in 7/10 (70%) versus 3/5 (60%) volunteers, respectively. Furthermore, specific antibody levels and cellular immune memory responses were comparable between both groups. We therefore conclude that mefloquine and chloroquine are equally effective in CPS-induced immune responses and protection. Trial registration: ClinicalTrials.gov NCT01422954.