2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

PK/PD and Bioanalytical Considerations of AAV-Based Gene Therapies: an IQ Consortium Industry Position Paper.

Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.

Nonclinical pharmacokinetics and biodistribution of VSV-GP using methods to decouple input drug disposition and viral replication.

Viral replication places oncolytic viruses (OVs) in a unique niche in the field of drug pharmacokinetics (PK) as their self-amplification obscures exposure-response relationships. Moreover, standard bioanalytical techniques are unable to distinguish the input from replicated drug products. Here, we combine two novel approaches to characterize PK and biodistribution (BD) after systemic administration of vesicular stomatitis virus pseudotyped with lymphocytic choriomeningitis virus glycoprotein (VSV-GP) in healthy mice. First: to decouple input drug PK/BD versus replication PK/BD, we developed and fully characterized a replication-incompetent tool virus that retained all other critical attributes of the drug. We used this approach to quantify replication in blood and tissues and to determine its impact on PK and BD. Second: to discriminate the genomic and antigenomic viral RNA strands contributing to replication dynamics in tissues, we developed an in situ hybridization method using strand-specific probes and assessed their spatiotemporal distribution in tissues. This latter approach demonstrated that distribution, transcription, and replication localized to tissue-resident macrophages, indicating their role in PK and BD. Ultimately, our study results in a refined PK/BD profile for a replicating OV, new proposed PK parameters, and deeper understanding of OV PK/BD using unique approaches that could be applied to other replicating vectors.

Assessment of the Immunogenicity Potential for Oligonucleotide-Based Drugs.

Therapeutic oligonucleotides (ONs) have characteristics of both small molecules and biologics. Although safety assessment of ONs largely follows guidelines established for small molecules, the unique characteristics of ONs often require incorporation of concepts from the safety assessment of biologics. The assessment of immunogenicity for ON therapeutics is one area where the approach is distinct from either established small molecule or biologic platforms. Information regarding immunogenicity of ONs is limited, but indicates that administration of ONs can result in antidrug antibody formation. In this study, we summarize the collective experience of the Oligonucleotide Safety Working Group in designing the immunogenicity assessment appropriate for this class of therapeutic, including advice on assay development, clinical monitoring, and evaluation of the impact of immunogenicity on exposure, efficacy, and safety of therapeutic ONs.

Considerations and recommendations for assessment of plasma protein binding and drug-drug interactions for siRNA therapeutics.

At the time of writing, although siRNA therapeutics are approved for human use, no official regulatory guidance specific to this modality is available. In the absence of guidance, preclinical development for siRNA followed a hybrid of the small molecule and biologics guidance documents. However, siRNA differs significantly from small molecules and protein-based biologics in its physicochemical, absorption, distribution, metabolism and excretion properties, and its mechanism of action. Consequently, certain reports typically included in filing packages for small molecule or biologics may benefit from adaption, or even omission, from an siRNA filing. In this white paper, members of the 'siRNA working group' in the IQ Consortium compile a list of reports included in approved siRNA filing packages and discuss the relevance of two in vitro reports-the plasma protein binding evaluation and the drug-drug interaction risk assessment-to support siRNA regulatory filings. Publicly available siRNA approval packages and the literature were systematically reviewed to examine the role of siRNA plasma protein binding and drug-drug interactions in understanding pharmacokinetic/pharmacodynamic relationships, safety and translation. The findings are summarized into two decision trees to help guide industry decide when in vitro siRNA plasma protein binding and drug-drug interaction studies are warranted.

Airway Delivery of Anti-influenza Monoclonal Antibodies Results in Enhanced Antiviral Activities and Enables Broad-Coverage Combination Therapies.

Effective and reliable anti-influenza treatments are acutely needed and passive immunizations using broadly neutralizing anti-influenza monoclonal antibodies (bNAbs) are a promising emerging approach. Because influenza infections are initiated in and localized to the pulmonary tract, and newly formed viral particles egress from the apical side of the lung epithelium, we compared the effectiveness of hemagglutinin (HA) stalk-binding bNAbs administered through the airway (intranasal or via nebulization) versus the systemic route (intraperitoneal or intravenous). Airway deliveries of various bNAbs were 10- to 50-fold more effective than systemic deliveries of the same bNAbs in treating H1N1, H3N2, B/Victoria-, and B/Yamagata-lineage influenza viral infections in mouse models. The potency of airway-delivered anti-HA bNAbs was highly dependent on antiviral neutralization activity, with little dependence on the effector function of the antibody. In contrast, the effectiveness of systemically delivered anti-HA bNAbs was not dependent on antiviral neutralization, but critically dependent on antibody effector functions. Concurrent administration of a neutralizing/effector function-positive bNAb via the airway and systemic routes showed increased effectiveness. The small amount of airway-delivered bNAbs needed for effective influenza treatment creates the opportunity to combine potent bNAbs with different anti-influenza specificities to generate a cost-effective antiviral therapy that provides broad coverage against all circulating influenza strains infecting humans. A 3 mg/kg dose of the novel triple antibody combination CF-404 (i.e., 1 mg/kg of each component bNAb) delivered to the airway was shown to effectively prevent weight loss and death in mice challenged with ten 50% lethal dose (LD50) inoculums of either H1N1, H3N2, B/Victoria-lineage, or B/Yamagata-lineage influenza viruses.IMPORTANCE Influenza causes widespread illness in humans and can result in morbidity and death, especially in the very young and elderly populations. Because influenza vaccination can be poorly effective some years, and the immune systems of the most susceptible populations are often compromised, passive immunization treatments using broadly neutralizing antibodies is a promising therapeutic approach. However, large amounts of a single antibody are required for effectiveness when delivered through systemic administration (typically intravenous infusion), precluding the feasible dosing of antibody combinations via this route. The significance of our research is the demonstration that effective therapeutic treatments of multiple relevant influenza types (H1N1, H3N2, and B) can be achieved by airway administration of a single combination of relatively small amounts of three anti-influenza antibodies. This advance exploits the discovery that airway delivery is a more potent way of administering anti-influenza antibodies compared to systemic delivery, making this a feasible and cost-effective therapeutic approach.

Chemokine Receptor 7 Is Essential for Coxiella burnetii Whole-Cell Vaccine-Induced Cellular Immunity but Dispensable for Vaccine-Mediated Protective Immunity.

BACKGROUND: Protective immunity against Coxiella burnetii infection is conferred by vaccination with virulent (PI-WCV), but not avirulent (PII-WCV) whole-cell inactivated bacterium. The only well-characterized antigenic difference between virulent and avirulent C. burnetii is they have smooth and rough lipopolysaccharide (LPS), respectively. METHODS: Mice were vaccinated with PI-WCV and PII-WCV. Humoral and cellular responses were evaluated using protein chip microarrays and ELISpots, respectively. Dendritic cell (DC) maturation after stimulation with PI-WVC and PII-WVC was evaluated using flow cytometry. Vaccine-challenge studies were performed to validate the importance of the receptor CCR7. RESULTS: Other than specific antibody response to PI-LPS, similar antibody profiles were observed but IgG titers were significantly higher after vaccination with PI-WCV. Furthermore, higher frequency of antigen-specific CD4+ T cells was detected in mice immunized with PI-WCV. PI-WCV-stimulated DCs displayed significantly higher levels of CCR7 and migratory ability to secondary lymphoid organs. Challenge-protection studies in wild-type and CCR7-deficient mice confirmed that CCR7 is critical for PI-WCV-induced cellular immunity. CONCLUSIONS: PI-WVC stimulates protective immunity to C. burnetii in mice through stimulation of migratory behavior in DCs for protective cellular immunity. Additionally, the humoral immune response to LPS is an important component of protective immunity.

Native Human Monoclonal Antibodies with Potent Cross-Lineage Neutralization of Influenza B Viruses.

Although antibodies that effectively neutralize a broad set of influenza viruses exist in the human antibody repertoire, they are rare. We used a single-cell screening technology to identify rare monoclonal antibodies (MAbs) that recognized a broad set of influenza B viruses (IBV). The screen yielded 23 MAbs with diverse germ line origins that recognized hemagglutinins (HAs) derived from influenza strains of both the Yamagata and Victoria lineages of IBV. Of the 23 MAbs, 3 exhibited low expression in a transient-transfection system, 4 were neutralizers that bound to the HA head region, 11 were stalk-binding nonneutralizers, and 5 were stalk-binding neutralizers, with 4 of these 5 having unique antibody sequences. Of these four unique stalk-binding neutralizing MAbs, all were broadly reactive and neutralizing against a panel of multiple strains spanning both IBV lineages as well as highly effective in treating lethal IBV infections in mice at both 24 and 72 h postinfection. The MAbs in this group were thermostable and bound different epitopes in the highly conserved HA stalk region. These characteristics suggest that these MAbs are suitable for consideration as candidates for clinical studies to address their effectiveness in the treatment of IBV-infected patients.

Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis.

BACKGROUND: Cytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development. METHODS: Recent sequencing of the C. felis genome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felis proteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model. RESULTS: Probing of the protein microarray resulted in identification of 30 differentially reactive C. felis antigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis. CONCLUSIONS: Protein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felis antigens. These antigens should be considered for development of C. felis vaccines, diagnostics, and therapeutics.

Measurements of Monoclonal Antibody Self-Association Are Correlated with Complex Biophysical Properties.

Successful development of monoclonal antibodies (mAbs) for therapeutic applications requires identification of mAbs with favorable biophysical properties (high solubility and low viscosity) in addition to potent bioactivities. Nevertheless, mAbs can also display complex, nonconventional biophysical properties that impede their development such as formation of soluble aggregates and subvisible particles as well as nonspecific interactions with various types of surfaces such as nonadsorptive chromatography columns. Here we have investigated the potential of using antibody self-interaction measurements obtained via affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) at dilute concentrations (0.01 mg/mL) for ranking a panel of 12 mAbs in terms of their expected biophysical properties at higher concentrations (1-30 mg/mL). Several mAb properties (solubility, % monomer, size-exclusion elution time and % recovery) displayed modest correlation with each other, as some mAbs with deficiencies in one or more properties (e.g., solubility) failed to show deficiencies in other properties (e.g., % monomer). The ranking of mAbs in terms of their level of self-association was correlated with their solubility ranking. However, the correlation was even stronger between the average ranking of the four biophysical properties and the AC-SINS measurements. This finding suggests that weak self-interactions detected via AC-SINS can manifest themselves in different ways and lead to complex biophysical properties. Our findings highlight the potential for using high-throughput self-interaction measurements to improve the identification of mAbs that possess a collection of excellent biophysical properties without the need for cumbersome analysis of each individual property during early candidate selection.

Identification of seroreactive proteins of Leptospira interrogans serovar copenhageni using a high-density protein microarray approach.

BACKGROUND: Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease. METHODOLOGY: In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results. PRINCIPAL FINDINGS: We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients. CONCLUSIONS: Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for diagnosing this neglected tropical disease. Further research is needed to assess the utility of these antigens in more deployable diagnostic platforms.

A novel candidate vaccine for cytauxzoonosis inferred from comparative apicomplexan genomics.

Cytauxzoonosis is an emerging infectious disease of domestic cats (Felis catus) caused by the apicomplexan protozoan parasite Cytauxzoon felis. The growing epidemic, with its high morbidity and mortality points to the need for a protective vaccine against cytauxzoonosis. Unfortunately, the causative agent has yet to be cultured continuously in vitro, rendering traditional vaccine development approaches beyond reach. Here we report the use of comparative genomics to computationally and experimentally interpret the C. felis genome to identify a novel candidate vaccine antigen for cytauxzoonosis. As a starting point we sequenced, assembled, and annotated the C. felis genome and the proteins it encodes. Whole genome alignment revealed considerable conserved synteny with other apicomplexans. In particular, alignments with the bovine parasite Theileria parva revealed that a C. felis gene, cf76, is syntenic to p67 (the leading vaccine candidate for bovine theileriosis), despite a lack of significant sequence similarity. Recombinant subdomains of cf76 were challenged with survivor-cat antiserum and found to be highly seroreactive. Comparison of eleven geographically diverse samples from the south-central and southeastern USA demonstrated 91-100% amino acid sequence identity across cf76, including a high level of conservation in an immunogenic 226 amino acid (24 kDa) carboxyl terminal domain. Using in situ hybridization, transcription of cf76 was documented in the schizogenous stage of parasite replication, the life stage that is believed to be the most important for development of a protective immune response. Collectively, these data point to identification of the first potential vaccine candidate antigen for cytauxzoonosis. Further, our bioinformatic approach emphasizes the use of comparative genomics as an accelerated path to developing vaccines against experimentally intractable pathogens.

Distinct human antibody response to the biological warfare agent Burkholderia mallei.

The genetic similarity between Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis) had led to the general assumption that pathogenesis of each bacterium would be similar. In 2000, the first human case of glanders in North America since 1945 was reported in a microbiology laboratory worker. Leveraging the availability of pre-exposure sera for this individual and employing the same well-characterized protein array platform that has been previously used to study a large cohort of melioidosis patients in southeast Asia, we describe the antibody response in a human with glanders. Analysis of 156 peptides present on the array revealed antibodies against 17 peptides with a > 2-fold increase in this infection. Unexpectedly, when the glanders data were compared with a previous data set from B. pseudomallei infections, there were only two highly increased antibodies shared between these two infections. These findings have implications in the diagnosis and treatment of B. mallei and B. pseudomallei infections.

Humoral immune responses to Plasmodium falciparum among HIV-1-infected Kenyan adults.

INTRODUCTION: Humoral immune responses play a pivotal role in naturally acquired immunity to malaria. Understanding which humoral responses are impaired among individuals at higher risk for malaria may improve our understanding of malaria immune control and contribute to vaccine development. METHODS: We compared humoral responses with 483 Plasmodium falciparum antigens between adults in, Kisumu (high, year-long malaria transmission leading to partial immunity), and adults in Kisii (low, seasonal malaria transmission). Then within each site, we compared malaria-specific humoral responses between those at higher risk for malaria (CD4(+) ≤500) and those at lower risk for malaria (CD4(+) >500). A protein microarray chip containing 483 P. falciparum antigens and 71 HIV antigens was used. Benjamini-Hochberg adjustments were made to control for multiple comparisons. RESULTS: Fifty-seven antigens including CSP, MSP1, LSA1 and AMA1 were identified as significantly more reactive in Kisumu than in Kisii. Ten of these antigens had been identified as protective in an earlier study. CD4(+) T-cell count did not significantly impact humoral responses. CONCLUSION: Protein microarrays are a useful method to screen multiple humoral responses simultaneously. This study provides useful clues for potential vaccine candidates. Modest decreases in CD4 counts may not significantly impact malaria-specific humoral immunity.

Profiling the humoral immune response of acute and chronic Q fever by protein microarray.

Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.

Identification of immunodominant antigens by probing a whole Chlamydia trachomatis open reading frame proteome microarray using sera from immunized mice.

Chlamydia trachomatis infections can lead to severe chronic complications, including trachoma, ectopic pregnancy, and infertility. The only effective approach to disease control is vaccination. The goal of this work was to identify new potential vaccine candidates through a proteomics approach. We constructed a protein chip array (Antigen Discovery, Inc.) by expressing the open reading frames (ORFs) from C. trachomatis mouse pneumonitis (MoPn) genomic and plasmid DNA and tested it with serum samples from MoPn-immunized mice. Two groups of BALB/c female mice were immunized either intranasally or intravaginally with live elementary bodies (EB). Another two groups were immunized by a combination of the intramuscular and subcutaneous routes with UV-treated EB (UV-EB), using either CpG and Montanide as adjuvants to favor a Th1 response or alum to elicit a Th2 response. Serum samples collected at regular intervals postimmunization were tested in the proteome array. The microarray included the expression products of 909 proteins from a total of 921 ORFs of the Chlamydia MoPn genome and plasmid. A total of 185 immunodominant proteins elicited an early and sustained antibody response in the mice immunized with live EB, and of these, 71 were also recognized by the sera from mice immunized with UV-EB. The reactive antigens included some proteins that were previously described as immunogenic, such as the major outer membrane protein, OmpB, Hsp60, and IncA and proteins from the type III secretion system. In addition, we identified in mice several new immunogens, including 75 hypothetical proteins. In summary, we have identified a new group of immunodominant chlamydial proteins that can be tested for their ability to induce protection.

High-throughput prediction of protein antigenicity using protein microarray data.

MOTIVATION: Discovery of novel protective antigens is fundamental to the development of vaccines for existing and emerging pathogens. Most computational methods for predicting protein antigenicity rely directly on homology with previously characterized protective antigens; however, homology-based methods will fail to discover truly novel protective antigens. Thus, there is a significant need for homology-free methods capable of screening entire proteomes for the antigens most likely to generate a protective humoral immune response. RESULTS: Here we begin by curating two types of positive data: (i) antigens that elicit a strong antibody response in protected individuals but not in unprotected individuals, using human immunoglobulin reactivity data obtained from protein microarray analyses; and (ii) known protective antigens from the literature. The resulting datasets are used to train a sequence-based prediction model, ANTIGENpro, to predict the likelihood that a protein is a protective antigen. ANTIGENpro correctly classifies 82% of the known protective antigens when trained using only the protein microarray datasets. The accuracy on the combined dataset is estimated at 76% by cross-validation experiments. Finally, ANTIGENpro performs well when evaluated on an external pathogen proteome for which protein microarray data were obtained after the initial development of ANTIGENpro. AVAILABILITY: ANTIGENpro is integrated in the SCRATCH suite of predictors available at http://scratch.proteomics.ics.uci.edu. CONTACT: pfbaldi@ics.uci.edu

Identification of the feline humoral immune response to Bartonella henselae infection by protein microarray.

BACKGROUND: Bartonella henselae is the zoonotic agent of cat scratch disease and causes potentially fatal infections in immunocompromised patients. Understanding the complex interactions between the host's immune system and bacterial pathogens is central to the field of infectious diseases and to the development of effective diagnostics and vaccines. METHODOLOGY: We report the development of a microarray comprised of proteins expressed from 96% (1433/1493) of the predicted ORFs encoded by the genome of the zoonotic pathogen Bartonella henselae. The array was probed with a collection of 62 uninfected, 62 infected, and 8 "specific-pathogen free" naïve cat sera, to profile the antibody repertoire elicited during natural Bartonella henselae infection. CONCLUSIONS: We found that 7.3% of the B. henselae proteins on the microarray were seroreactive and that seroreactivity was not evenly distributed between predicted protein function or subcellular localization. Membrane proteins were significantly most likely to be seroreactive, although only 23% of the membrane proteins were reactive. Conversely, we found that proteins involved in amino acid transport and metabolism were significantly underrepresented and did not contain any seroreactive antigens. Of all seroreactive antigens, 52 were differentially reactive with sera from infected cats, and 53 were equally reactive with sera from infected and uninfected cats. Thirteen of the seroreactive antigens were found to be differentially seroreactive between B. henselae type I and type II. Based on these results, we developed a classifier algorithm that was capable of accurately discerning 93% of the infected animals using the microarray platform. The seroreactivity and diagnostic potential of these antigens was then validated on an immunostrip platform, which correctly identified 98% of the infected cats. Our protein microarray platform provides a high-throughput, comprehensive analysis of the feline humoral immune response to natural infection with the alpha-proteobacterium B. henselae at an antigen-specific, sera-specific, and genome-wide level. Furthermore, these results provide novel insight and utility in diagnostics, vaccine development, and understanding of host-pathogen interaction.

Genome-wide profiling of humoral immune response to Coxiella burnetii infection by protein microarray.

Comprehensive evaluation of the humoral immune response to Coxiella burnetii may identify highly needed diagnostic antigens and potential subunit vaccine candidates. Here we report the construction of a protein microarray containing 1901 C. burnetii ORFs (84% of the entire proteome). This array was probed with Q-fever patient sera and naïve controls in order to discover C. burnetii-specific seroreactive antigens. Among the 21 seroreactive antigens identified, 13 were significantly more reactive in Q-fever cases than naïve controls. The remaining eight antigens were cross-reactive in both C. burnetii infected and naïve patient sera. An additional 64 antigens displayed variable seroreactivity in Q-fever patients, and underscore the diversity of the humoral immune response to C. burnetii. Nine of the differentially reactive antigens were validated on an alternative immunostrip platform, demonstrating proof-of-concept development of a consistent, safe, and inexpensive diagnostic assay alternative. Furthermore, we report here the identification of several new diagnostic antigens and potential subunit vaccine candidates for the highly infectious category B alphaproteobacteria, C. burnetii.

Defining the humoral immune response to infectious agents using high-density protein microarrays.

A major component of the adaptive immune response to infection is the generation of protective and long-lasting humoral immunity. Traditional approaches to understanding the host's humoral immune response are unable to provide an integrated understanding of the antibody repertoire generated in response to infection. By studying multiple antigenic responses in parallel, we can learn more about the breadth and dynamics of the antibody response to infection. Measurement of antibody production following vaccination is also a gauge for efficacy, as generation of antibodies can protect from future infections and limit disease. Protein microarrays are well suited to identify, quantify and compare individual antigenic responses following exposure to infectious agents. This technology can be applied to the development of improved serodiagnostic tests, discovery of subunit vaccine antigen candidates, epidemiologic research and vaccine development, as well as providing novel insights into infectious disease and the immune system. In this review, we will discuss the use of protein microarrays as a powerful tool to define the humoral immune response to bacteria and viruses.