Candidate Antigens Research Articles

Immunogenicity and protective efficacy of a multi-epitope recombinant toxin antigen of Pasteurella multocida against virulent challenge in mice.

Pasteurella multocida (P. multocida) infection frequently results in porcine atrophic rhinitis and swine plague, leading to large economic losses for the swine industry worldwide. P. multocida toxin (PMT, 146kDa) is a highly virulent key virulence factor that plays a vital role in causing lung and turbinate lesions. This study developed a multi-epitope recombinant antigen of PMT (rPMT) that showed excellent immunogenicity and protection in a mouse model. Using bioinformatics to analyse the dominant epitopes of PMT, we constructed and synthesized rPMT containing 10 B-cell epitopes, 8 peptides with multiple B-cell epitopes and 13 T-cell epitopes of PMT and a rpmt gene (1,974bp) with multiple epitopes. The rPMT protein (97kDa) was soluble and contained a GST tag protein. Immunization of mice with rPMT stimulated significantly elevated serum IgG titres and splenocyte proliferation, and serum IFN-γ and IL-12 were upregulated by 5-fold and 1.6-fold, respectively, but IL-4 was not. Furthermore, the rPMT immunization group exhibited alleviated lung tissue lesions and a significantly decreased degree of neutrophil infiltration compared with the control groups post-challenge. In the rPMT vaccination group, 57.1% (8/14) of the mice survived the challenge, similar to the bacterin HN06 group, while all the mice in the control groups succumbed to the challenge. Thus, rPMT could be a suitable candidate antigen for developing a subunit vaccine against toxigenic P. multocida infection.

Immunoinformatics and tick vaccinology

Immunoinformatics is an emerging area focused on development and applications of methods used to facilitate vaccine development. There is a growing interest in the field of vaccinology centered on the new omic science named ‘vaccinomics’. However, this approach has not succeeded to provide a solution against major infections affecting both animals and humans, since tick vaccines are still being developed based on conventional biochemical or immunological methods to dissect the molecular structure of the pathogen, looking for a candidate antigen. The availability of complete genomes and the novel advanced technologies, such as data mining, bioinformatics, microarrays, and proteomics, have revolutionized the approach to vaccine development and provided a new impulse to tick research. The aim of this review is to explore how modern vaccinology will contribute to the discovery of new candidate antigens and to understand the research process to improve existing vaccines. Under this concept, the omic age of ticks will make it possible to design vaccines starting from a prediction based on the in silico analysis of gene sequences obtained by data mining using computer algorithms, without the need to keep the pathogen growing in vitro. This new genome-based approach has been named “reverse vaccinology 3.0” or “vaccinomics 1.0” and can be applied to ticks.

Antigenicity and immunogenicity of SARS-CoV-2 surface glycoprotein fragment in CHO cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein that projects from the virus surface is highly immunogenic. It is considered to be the target of many neutralizing antibodies as well as a target in vaccine design efforts. Evaluation the immunogenicity of a recombinant fragment of the spike protein (rfsp) that is comprised of Receptor Binding Domain (RBD), S1/S2 cleavage site, and fusion peptide (FP) as immunogenic proteins of SARS-COV-2, in BALB/c mice and evaluation of the efficacy of epitopes rfsp as a multi-subunit chimeric vaccine. The present study made use of CHO-K1 (Chinese hamster ovary K1) cells to create a cell line for constant expression rfsp. The rfsp was purified with Ni-NTA chromatography and confirmed by Western blotting. The immunogenicity and neutralizing antibody efficacy of rfsp were evaluated in BALB/c mice. ELISA was employed to test rfsp via sera of COVID-19 convalescent patients infected with SARS-CoV-2 alpha and delta variants. Our results showed significant differences in antibody titers in immunized mice compared to the control groups and neutralizing antibodies were positive, sera from mice immunized are capable of bound SARS-CoV-2 virus, chimer peptide is capable bound antibodies patients infected with SARS-CoV-2 and patients infected with delta variant SARS-CoV-2. Overall, these results indicate that rfsp protein would be a novel potential antigen candidate for the development of a subunit SARS CoV-2 vaccine and rfsp has the potential to be a useful option for the development of the assays for serodiagnosis of SARS-CoV-2 infection.

Preparation of polyclonal anti-Schistosoma mansoni cysteine protease antibodies for early diagnosis

In many parts of the tropics, schistosomiasis is a major parasitic disease second only to malaria as a cause of morbidity and mortality. Diagnostic approaches include microscopic sampling of excreta such as the Kato-Katz method, radiography, and serology. Due to their vital role in many stages of the parasitic life cycle, proteases have been under investigation as targets of immunological or chemotherapeutic anti-Schistosoma agents. Five major classes of protease have been identified on the basis of the peptide hydrolysis mechanism: serine, cysteine, aspartic, threonine, and metalloproteases. Proteases of all five catalytic classes have been identified from S. mansoni through proteomic or genetic analysis. The study aimed to produce polyclonal antibodies (pAbs) against schistosomal cysteine proteases (CP) to be used in the diagnosis of schistosomiasis. This study was conducted on S. mansoni-infected patients from highly endemic areas and from outpatients’ clinic and hospitals and other patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids). In this study, the produced polyclonal antibodies against S. mansoni cysteine protease antigens were labeled with horseradish peroxidase (HRP) conjugate and used to detect CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA. The study involved 200 S. mansoni-infected patients (diagnosed by finding characteristic eggs in the collected stool samples), 100 patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids), and 100 individuals who served as parasite-free healthy negative control. The prepared pAb succeeded in detecting CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA with a sensitivity of 98.5% and 98.0% respectively. A positive correlation was observed between S. mansoni egg counts and both stool and serum antigen concentrations. Purified 27.5 kDa CP could be introduced as a suitable candidate antigen for early immunodiagnosis using sandwich ELISA for antigen detection.Key points• Detection of cysteine protease antigens can replace parasitological examination.• Sandwich ELISA has a higher sensitivity than microscopic examination of eggs.• Identification of antigens is important for the goal of obtaining diagnostic tools.

Streptococcus pyogenes vaccine candidates do not induce autoimmune responses in a rheumatic heart disease model

We have developed a candidate vaccine to protect against multiple strains of Streptococcus pyogenes infections. The candidate vaccine contains two synthetic peptides derived from S. pyogenes proteins: the M-protein epitope, p*17 and the IL-8 degrading S. pyogenes Cell-Envelope Proteinase (SpyCEP) epitope, K4S2. In this study we utilise a rat autoimmune valvulitis model that displays both the cardiac and neurobehavioural pathology associated with post-streptococcal sequelae, to assess if the vaccine candidate antigens induce autoimmune complications and inflammatory pathology. Each antigen was conjugated to carrier protein diphtheria toxoid (DT) and independently assessed for potential to induce autoimmune pathology in female Lewis rats. Rats were administered three subcutaneous doses, and one intranasal dose over a four-week study with a two-week recovery period. A positive control group received recombinant S. pyogenes M5 (rM5) protein, and the negative control group received PBS. Rats that received rM5 developed significant cardiac and neurological pathologies. There was no evidence of these pathologies in the PBS control group, or the rats administered either P*17-DT or K4S2-DT. This study provides further preclinical evidence of the safety of the vaccine candidates p*17 and K4S2 and their appropriateness as candidates in human clinical trials.

Characterization and expression of domains of Alphaherpesvirus bovine 1/5 envelope glycoproteins B in Komagataella phaffi

BackgroundBovine herpes virus (BoHV 1 and BoHV-5) are the causative agents of infectious bovine rhinotracheitis (IBR). IBR is responsible for important economic losses in the cattle industry. The envelope glycoprotein B (gB) is essential for BoHV infection of cattle's upper respiratory and genital tract. gB is one of the main candidate antigens for a potential recombinant vaccine since it induces a strong and persistent immune response.ResultsIn this study, gB of BoHV-1 and BoHV-5 was characterized in terms of function, structure, and antigenicity through bioinformatics tools. gB showed conserved sequence and structure, so, both domains named PH Like 1 and 2 domains of each virus were selected for the design of a bivalent vaccine candidate. The immunoinformatic study showed that these two domains have epitopes recognizable by B and T lymphocytes, followed by this, the cDNA domains from BoHV-1/5 gB (Domains-gB) were transformed into the yeast Komagataella phaffii GS115 (previously known as Pichia pastoris). A recombinant protein with molecular weight of about 110 kDa was obtained from the culture media. The vaccine candidate protein (Domains-gB) was recognized by a monoclonal antibody from a commercial ELISA kit used for IBR diagnostic, which may suggest that the epitopes are conserved of the entire infectious virus.ConclusionOverall, it was shown that the recombinant domains of BoHV-1/5 gB have antigenic and immunogenic properties similar to the native gB. This vaccine candidate is promising to be used in future studies to assess its immunogenicity in an animal model.

Evolution of Tick Vaccinology Highlights Changes in Paradigms in This Research Area.

Ticks and tick-borne diseases affect human and animal health worldwide. Although some tick-protective antigens have been identified and characterized, further research is needed for the development and application of effective anti-tick vaccines, which currently are unavailable for human protection. To study the trends and gaps in anti-tick vaccine approaches, herein we used scientometric analysis to evaluate several aspects of tick vaccinology. Co-authorship and citations networks pointed out two main research fronts, one focused on the laboratory protocols driving the recognition of candidate antigens and the other devoted to field experiments of protection against ticks. The analysis demonstrated the prominence of research in European countries on the topic. The scientometric approach allowed the recognition of isolated teams working casually on the topic, the lack of cooperation between middle- and low-income countries, and the need for sustained and integrated research. Most important, we identified a considerable lack of new candidates for vaccine development, as well as the participation of African and Asian countries. These results provide significant insights obtained from bibliographical analysis, suggest the strength and weaknesses in this field of research, and highlight new directions to advance in the development of effective vaccines for the control of tick infestations and tick-borne pathogens.

In Vivo Gene Expression Profiling of Staphylococcus aureus during Infection Informs Design of Stemless Leukocidins LukE and -D as Detoxified Vaccine Candidates.

Staphylococcus aureus is a clinically important bacterial pathogen that has become resistant to treatment with most routinely used antibiotics. Alternative strategies, such as vaccination and phage therapy, are therefore actively being investigated to prevent or combat staphylococcal infections. Vaccination requires that vaccine targets are expressed at sufficient quantities during infection so that they can be targeted by the host's immune system. While our knowledge of in vitro expression levels of putative vaccine candidates is comprehensive, crucial in vivo expression data are scarce and promising vaccine candidates during in vitro assessment often prove ineffective in preventing S. aureus infection. Here, we show how a newly developed high-throughput quantitative reverse transcription-PCR (qRT-PCR) assay monitoring the expression of 84 staphylococcal genes encoding mostly virulence factors can inform the selection and design of effective vaccine candidates against staphylococcal infections. We show that this assay can accurately quantify mRNA expression levels of these genes in several host organs relying only on very limited amounts of bacterial mRNA in each sample. We selected two highly expressed genes, lukE and lukD, encoding pore-forming leukotoxins, to inform the design of detoxified recombinant proteins and showed that immunization with recombinant genetically detoxified LukED antigens conferred protection against staphylococcal skin infection in mice. Consequently, knowledge of in vivo-expressed virulence determinants can be successfully deployed to identify and select promising candidates for optimized design of effective vaccine antigens against S. aureus. Notably, this approach should be broadly applicable to numerous other pathogens. IMPORTANCE Vaccination is an attractive strategy for preventing bacterial infections in an age of increased antimicrobial resistance. However, vaccine development frequently suffers significant setbacks when candidate antigens that show promising results in in vitro experimentation fail to protect from disease. An alluring strategy is to focus resources on developing bacterial virulence factors that are expressed during disease establishment or maintenance and are critical for bacterial in-host survival as vaccine targets. While expression profiles of many virulence factors have been characterized in detail in vitro, our knowledge of their in vivo expression profiles is still scarce. Here, using a high-throughput qRT-PCR approach, we identified two highly expressed leukotoxins in a murine infection model and showed that genetically detoxified derivatives of these elicited a protective immune response in a murine skin infection model. Therefore, in vivo gene expression can inform the selection of promising candidates for the design of effective vaccine antigens.

Plasmodium vivax vaccine: What is the best way to go?

Malaria is one of the most devastating human infectious diseases caused by Plasmodium spp. parasites. A search for an effective and safe vaccine is the main challenge for its eradication. Plasmodium vivax is the second most prevalent Plasmodium species and the most geographically distributed parasite and has been neglected for decades. This has a massive gap in knowledge and consequently in the development of vaccines. The most significant difficulties in obtaining a vaccine against P. vivax are the high genetic diversity and the extremely complex life cycle. Due to its complexity, studies have evaluated P. vivax antigens from different stages as potential targets for an effective vaccine. Therefore, the main vaccine candidates are grouped into preerythrocytic stage vaccines, blood-stage vaccines, and transmission-blocking vaccines. This review aims to support future investigations by presenting the main findings of vivax malaria vaccines to date. There are only a few P. vivax vaccines in clinical trials, and thus far, the best protective efficacy was a vaccine formulated with synthetic peptide from a circumsporozoite protein and Montanide ISA-51 as an adjuvant with 54.5% efficacy in a phase IIa study. In addition, the majority of P. vivax antigen candidates are polymorphic, induce strain-specific and heterogeneous immunity and provide only partial protection. Nevertheless, immunization with recombinant proteins and multiantigen vaccines have shown promising results and have emerged as excellent strategies. However, more studies are necessary to assess the ideal vaccine combination and test it in clinical trials. Developing a safe and effective vaccine against vivax malaria is essential for controlling and eliminating the disease. Therefore, it is necessary to determine what is already known to propose and identify new candidates.

Anti-LGI4 Antibody Is a Novel Juxtaparanodal Autoantibody for Chronic Inflammatory Demyelinating Polyneuropathy.

The objective of this study was to discover novel nodal autoantibodies in chronic inflammatory demyelinating polyneuropathy (CIDP). We screened for autoantibodies that bind to mouse sciatic nerves and dorsal root ganglia (DRG) using indirect immunofluorescence (IFA) assays with sera from 113 patients with CIDP seronegative for anti-neurofascin 155 and anticontactin-1 antibodies and 127 controls. Western blotting, IFA assays using HEK293T cells transfected with relevant antigen expression plasmids, and cell-based RNA interference assays were used to identify target antigens. Krox20 and Periaxin expression, both of which independently control peripheral nerve myelination, was assessed by quantitative real-time PCR after application of patient and control sera to Schwann cells. Sera from 4 patients with CIDP, but not control sera, selectively bound to the nodal regions of sciatic nerves and DRG satellite glia (p = 0.048). The main immunoglobulin G (IgG) subtype was IgG4. IgG from these 4 patients stained a 60-kDa band on Western blots of mouse DRG and sciatic nerve lysates. These features indicated leucine-rich repeat LGI family member 4 (LGI4) as a candidate antigen. A commercial anti-LGI4 antibody and IgG from all 4 seropositive patients with CIDP showed the same immunostaining patterns of DRG and cultured rat Schwann cells and bound to the 60-kDa protein in Western blots of LGI4 overexpression lysates. IgG from 3 seropositive patients, but none from controls, bound to cells cotransfected with plasmids containing LGI4 and a disintegrin and metalloprotease domain-containing protein 22 (ADAM22), an LGI4 receptor. In cultured rat Schwann and human melanoma cells constitutively expressing LGI4, LGI4 siRNA effectively downregulated LGI4 and reduced patients' IgG binding compared with scrambled siRNA. Application of serum from a positive patient to Schwann cells expressing ADAM22 significantly reduced the expression of Krox20, but not Periaxin. Anti-LGI4 antibody-positive patients had a relatively old age at onset (mean age 58 years), motor weakness, deep and superficial sensory impairment with Romberg sign, and extremely high levels of CSF protein. Three patients showed subacute CIDP onset resembling Guillain-Barré syndrome. IgG4 anti-LGI4 antibodies are found in some elderly patients with CIDP who present subacute sensory impairment and motor weakness and are worth measuring, particularly in patients with symptoms resembling Guillain-Barré syndrome.

A comparative clinico-dermoscopic study of intralesional injection of combined digoxin and furosemide, Candida antigen, and vitamin D3 for multiple warts.

Immunostimulatory and antiproliferative therapies have been widely used for the treatment of multiple warts. Recently, anti-HPV activity of ionic contra viral therapy (ICVT) which is comprised of combined digoxin and furosemide has been demonstrated. To evaluate and compare the effectiveness and safety of intralesional injection of Candida antigen, vitamin D3, and combined digoxin and furosemide in the treatment of multiple warts. Seventy-five patients with numerous warts were randomly assigned to one of three equal groups: Candida antigen, vitamin D3, or a combination of digoxin and furosemide. In the Candida antigen group, injections into the biggest wart were done. In the vitamin D3 and combined digoxin/furosemide groups, the agent was injected into each wart with a maximum of five injected warts. Injections were repeated every 2 weeks until clearance or for a total of five sessions. There was a statistically significant difference in the overall therapeutic response among the studied groups in favor of the intralesional Candida antigen group (60%), followed by the vitamin D3 group (48%) and the ionic contraviral therapy group (28%) (p=0.02). However, the difference between both Candida antigen and vitamin D groups was not significant (p=0.59). Intralesional Candida antigen immunotherapy and vitamin D3 antiproliferative therapy are significantly more effective than ICVT. Short follow-up period and relatively small sample size.

Protective efficacy induced by Eimeria maxima rhomboid-like protein 1 against homologous infection.

Avian coccidiosis, caused by apicomplexan protozoa belonging to the Eimeria genus, is considered one of the most important diseases in the intensive poultry industry worldwide. Due to the shortcomings of live anticoccidial vaccines and drugs, the development of novel anticoccidial vaccines is increasingly urgent. Eimeria maxima rhomboid-like protein 1 (EmROM1), an invasion-related molecule, was selected as a candidate antigen to evaluate its protective efficacy against E. maxima in chickens. Firstly, the prokaryotic recombinant plasmid pET-32a-EmROM1 was constructed to prepare EmROM1 recombinant protein (rEmROM1), which was used as a subunit vaccine. The eukaryotic recombinant plasmid pVAX1.0-EmROM1 (pEmROM1) was constructed as a DNA vaccine. Subsequently, 2-week-old chicks were separately vaccinated with the rEmROM1 and pEmROM1 twice every 7 days. One week post the booster vaccination, induced cellular immune responses were determined by evaluating the mRNA level of cytokines including IL-2, IFN-γ, IL-4, IL-10, TGF-β, IL-17, and TNFSF15, as well as the percentages of CD4+ and CD8+ T cells from spleens of vaccinated chickens. Specific serum antibody level in the vaccinated chickens was determined to assess induced humoral immune responses. Finally, the protective efficacy of EmROM1 was evaluated by a vaccination-challenge trial. EmROM1 vaccination significantly upregulated the cytokine transcription levels and CD4+/CD8+ T cell percentages in vaccinated chickens compared with control groups, and also significantly increased the levels of serum-specific antibodies in vaccinated chickens. The animal trial showed that EmROM1 vaccination significantly reduced oocyst shedding, enteric lesions, and weight loss of infected birds compared with the controls. The anticoccidial index (ACI) from the rEmROM-vaccination group and pEmROM1-vaccination group were 174.11 and 163.37, respectively, showing moderate protection against E. maxima infection. EmROM1 is an effective candidate antigen for developing DNA or subunit vaccines against avian coccidiosis.

Retrospective analysis of treatment of cutaneous warts with measles, mumps, and rubella immunotherapy over 8 years

Introduction: Warts are benign lesions caused by human papilloma virus. Various types of cutaneous warts include verruca vulgaris, genital warts, and palmoplantar warts. Various therapeutic modalities are available for warts with varying response. These include destructive therapies, cytotoxic agents (Bleomycin), and immunotherapy (measles, mumps, and rubella [MMR], candida antigen, etc.). We have analyzed the efficacy of intralesional MMR immunotherapy in patients with different kinds of cutaneous warts. Aim: The purpose of this study was to retrospectively analyze the effectiveness and safety of MMR immunotherapy in the treatment of different kinds of cutaneous warts. Materials and Methods: We included all the patients with cutaneous warts receiving MMR vaccine between March 2014 and March 2022. Demographic data were recorded. MMR vaccine was given for four doses at 3 weeks interval or till there was complete clearance, whichever was earlier. Clearance and reduction of wart sizes and potential side effects were recorded. Results: A total of 184 patients were enrolled, and 45% patients were women. Predominant age group of patients was 21–40 years. Most common types of warts observed was palmoplantar warts. Complete resolution was seen in 66% patients and partial response in 22% patients. Palmo-plantar and warts on extremities responded completely to immunotherapy, whereas 43% of genital warts had no improvement. Pain at injection site was observed in all patients, and 32% patients had flu-like symptoms. Conclusions: Immunotherapy with MMR vaccine shows a promising response in the treatment of palmo-plantar warts and warts on extremities, without any serious adverse effect, whereas the genital warts and verruca plana respond variably to immunotherapy.

Recombinant Protein Mimicking the Antigenic Structure of the Viral Surface Envelope Protein Reinforces Induction of an Antigen-Specific and Virus-Neutralizing Immune Response Against Dengue Virus.

Dengue virus (DENV), belonging to the family Flaviviridae, is the causative agent of dengue and comprises four serotypes. A second heterologous DENV infection is a critical risk factor for severe dengue, and no effective vaccine is available to prevent infection by all four DENV serotypes. Recombinant DENV vaccines are primarily based on the envelope proteins, prM and E. The E protein and its envelope domain III (EDIII) have been investigated as candidate antigens (Ags) for recombinant subunit vaccines. However, most EDIII-based Ags are monomers that do not display the cognate antigenic structure of E protein, which is essential for induction of virus-neutralizing immunity. Here, we developed recombinant DENV-2 envelope domain (r2ED) protein as an Ag that mimics the quaternary structure of E protein on the DENV surface. We confirmed that r2ED retained the conformational epitope displayed at the E-dimer interface, which reportedly exhibits broad virus-neutralizing capacity, without displaying the fusion loop epitope that causes antibody (Ab)-dependent enhancement. Furthermore, compared with EDIII alone, r2ED elicited stronger Ag-specific and cross-reactive neutralizing Ab and T cell-mediated immune responses in mice. This Ag-specific immunity was maintained at an elevated level 6months after the last immunization, suggesting sustained Ag-specific immune memory. Taken together, these observations suggest that r2ED could be used to develop an improved subunit vaccine capable of inducing a broadly cross-reactive and long-lasting immune response against DENV infection.

Immunogenicity and Antigenicity of the Ectodomain of Rabies Virus Glycoprotein Stably Expressed in HEK293T Cells.

Rabies continues to be a huge threat to public health. The rabies virus envelope glycoprotein (RABV G) is a major rabies virus antigen and contains neutralizing epitopes, which are primary candidates for subunit vaccines and diagnostic antigens. However, the production and purification of rRABV G while retaining its antigenic and immunogenic remains to be a challenge. Here, we aimed to establish a platform for rRABV G production and purification, and determine the immunogenicity and antigenicity of rRABV G. The cDNA fragment encoding the soluble form of RABV G was synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-RABV G-eGFP was packaged, titered, and then transduced into HEK 293T cells. The cell culture supernatant was purified using nickel affinity chromatography and subsequently confirmed through Western Blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). The ELISA utilized human sera obtained from individuals who had been vaccinated with the human commercial Purified Vero Cells Rabies Vaccine (PVRV). Notably, we observed a neutralizing antibody response in immunized pigs rather than in mice. This discrepancy could potentially be attributed to factors such as the instability of the rRABV G protein, variations in host responses, and variances in the adjuvant used. Taking all these findings into account, the rRABV G protein generated in this study exhibits promise as a potential vaccine candidate for the prevention of rabies.

Potential of ferritin 2 as an antigen for the development of a universal vaccine for avian mites, poultry red mites, tropical fowl mites, and northern fowl mites.

Poultry red mites (PRMs, Dermanyssus gallinae), blood-sucking ectoparasites, are a threat to the poultry industry because of reduced production caused by infestation. In addition, tropical fowl mites (TFMs, Ornithonyssus bursa) and northern fowl mites (NFMs, Ornithonyssus sylviarum) are hematophagous, distributed in various regions, genetically and morphologically close to PRMs, and cause similar problems to the poultry industry. Vaccine approaches have been studied for PRM control, and several molecules have been identified in PRMs as candidates for effective vaccine antigens. The development of an anti-PRM vaccine as a universal vaccine with broad efficacy against avian mites could improve the productivity of poultry farms worldwide. Molecules that are highly conserved among avian mites and have critical functions in the physiology and growth of mites could be ideal antigen candidates for the development of universal vaccines. Ferritin 2 (FER2), an iron-binding protein, is critical for the reproduction and survival of PRMs and has been reported as a useful vaccine antigen for the control of PRMs and a candidate for the universal vaccine antigen in some tick species. Herein, we identified and characterized FER2 in TFMs and NFM. Compared with the sequence of PRM, the ferroxidase centers of the heavy chain subunits were conserved in FER2 of TFMs and NFMs. Phylogenetic analysis revealed that FER2 belongs to clusters of secretory ferritins of mites and other arthropods. Recombinant FER2 (rFER2) proteins from PRMs, TFMs, and NFMs exhibited iron-binding abilities. Immunization with each rFER2 induced strong antibody responses in chickens, and each immune plasma cross-reacted with rFER2 from different mites. Moreover, mortality rates of PRMs fed with immune plasma against rFER2 from TFMs or NFMs, in addition to PRMs, were higher than those of control plasma. rFER2 from each avian mite exhibited anti-PRM effects. This data suggests that it has the potential to be used as an antigen candidate for a universal vaccine against avian mites. Further studies are needed to access the usefulness of FER2 as a universal vaccine for the control of avian mites.

Detection of positive selection acting on protein surfaces at the whole-genome scale in the human malaria parasite Plasmodium falciparum.

The host-parasite evolutionary arms race is a fundamental process with medical implications. During this process, the host develops parasite resistance, and the parasite develops host immune evasion strategies. Thus, this process accelerates relevant protein evolution. This study test hypothesizes that proteins subject to sequence evolution structural constraints play a crucial role and that these constraints hinder the modification of such proteins in this process. These hypotheses were tested using Plasmodium falciparum model and evaluated protein structures predicted for the entire proteome by the AlphaFold method. Based on dN/dS test results and P. falciparum and P. reichenowi comparisons, the presented approach identified proteins subject to purifying selection acting on the whole sequence and buried residues (dN < dS) and positive selection on nonburied residues. Of the 26 proteins, some known antigens (ring-exported protein 3, RAP protein, erythrocyte binding antigen-140, and protein P47) targeted by the host immune system are promising vaccine candidates. The set also contained 11 enzymes, including FIKK kinase, which modifies host proteins. This set was compared with genes for which the dN/dS test suggested that positive selection acts on the whole gene (i.e., dN > dS). The present study found that such genes encode enzymes and antigenic vaccine candidates less frequently than genes for which evolution is not subject to selection constraints and positive selection acts on only exposed residues. The analysis was repeated comparing P. falciparum with P. alderi, which is more distantly related. The study discusses the potential implications of the presented methodology for rational vaccine design and the parasitology and evolutionary biology fields.

Vaccines against Cattle Ticks: Current Status

Rhipicephalus microplus, famous as the cattle tick, is a hematophagous ectoparasite distributed worldwide in tropical and subtropical regions. Its infestations represent a threat to the cattle industry since they generate a negative economic impact on cattle production and animal welfare. The method par excellence for tick control is based on applying chemical agents. However, their use has had limited efficacy, often accompanied by environmental contamination and the selection of tick’s resistant to most chemical agents, rendering them ineffective. One of the alternative methods to combat R. microplus infestations is the development of vaccines. Two commercial vaccines are based on the Bm86 antigen, TickGARD®, and Gavac®. However, the efficacy is highly variable between different geographical regions. While it is true that other alternatives have been tested, the search for vaccine antigens that provide high protection against R. microplus remains a challenge in which genomic, transcriptomic, and proteomic studies are required. This review briefly summarizes the current situation regarding the discovery of candidate antigens for vaccines against ticks in cattle, as well as the methodologies employed for their search and development.

CoV2-TCR: A web server for screening TCR CDR3 from TCR immune repertoire of COVID-19 patients and their recognized SARS-CoV-2 epitopes

Although multiple vaccines have been developed and widely administered, several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have been reported to evade immune responses and spread diffusely. Here, 108 RNA-seq files from coronavirus disease 2019 (COVID-19) patients and healthy donors (HD) were downloaded to extract their TCR immune repertoire by MiXCR. Those extracted TCR repertoire were compared and it was found that disease progression was related negatively with diversity and positively with clonality. Specifically, greater proportions of high-abundance clonotypes were observed in active and severe COVID-19 samples, probably resulting from strong stimulation of SARS-CoV-2 epitopes and a continued immune response in host. To investigate the specific recognition between TCR CDR3 and SARS-CoV-2 epitopes, we constructed an accurate classifier CoV2-TCR with an AUC of 0.967 in an independent dataset, which outperformed several similar tools. Based on this model, we observed a huge range in the number of those TCR CDR3 recognizing those different peptides, including 28 MHC-I epitopes from SARS-CoV-2 and 22 immunogenic peptides from SARS-CoV-2 variants. Interestingly, their proportions of high-abundance, low-abundance and rare clonotypes were close for each peptide. To expand the potential application of this model, we established the webserver, CoV2-TCR, in which users can obtain those recognizing CDR3 sequences from the TCR repertoire of COVID-19 patients based on the 9-mer peptides containing mutation site(s) on the four main proteins of SARS-CoV-2 variants. Overall, this study provides preliminary screening for candidate antigen epitopes and the TCR CDR3 that recognizes them, and should be helpful for vaccine design on SARS-CoV-2 variants.

Abstract IA016: Vaccine approach for the interception of colon cancer

Abstract This year, in the US, an estimated 150,000 people will be diagnosed with colon cancer and ~52,000 will die of the disease. A vaccine approach is well suited to colon cancer prevention. First, a limited number of immunizations can be given over a short period of time to achieve a desired immune response. If immunologic memory develops, antigen specific T-cells will persist in the body for years, ready to be deployed when aberrant cells are detected. Finally, there are several well-defined high-risk populations that would be suitable for testing a preventative colon cancer vaccine including individuals (1) with a hereditary genetic syndrome such as familial adenomatous polyposis, (2) with a history of inflammatory bowel disease such as Crohn’s disease or ulcerative colitis, and (3) those with a history of hyperplastic colonic polyps. Vaccines targeting virally mediated cancers are effective, in part, because T-cells are primed to proteins involved in oncogenesis. The most common type of colon adenoma and invasive cancer associated antigens are non-mutated growth-related proteins that become markedly overexpressed in the disease state. Genes which are aberrantly up-regulated and conserved from adenoma to invasive colon carcinoma may encode overexpressed proteins that are associated with colon cancer initiation. Evaluating gene expression data derived from over 600 colorectal cancers, matched normal tissues, and colon adenomas we identified 160 genes overexpressed in the majority of adenomas and early-stage colon cancers, but not in normal tissues. Using siRNA screening we demonstrated 35 of those genes, when silenced, resulted in apoptosis or inhibition of growth of colon cancer cells and an adenoma cell line but not a non-neoplastic colon cell line. Of those genes, 16 have been shown to encode proteins that are overexpressed in both adenomas and colon cancer and are associated with disease progression and over a third of these proteins are immunogenic. These proteins could serve as potential candidate antigens for inclusion in a multi-antigen vaccine. Recently, we have identified epitopes within the natural sequences of non-mutated tumor antigens that only generate Th1 (Th1 selective) antigen specific T-cells. This discovery and the development of high throughput methods to both identify and screen putative Class II binding peptides for Th selective function has allowed us to develop antigen specific Th1 selective vaccines. A vaccine targeting CDC25B, COX2, and EGFR can inhibit intestinal tumor formation in both APCmin mice and mice with tumor induced by AOM. Concomitant use of NSAIDs potentiates the immune response generated with vaccination. The anti-tumor response observed after vaccination is significantly correlated with elevated CD8 T-cells found in the lesions and induced by immunization as well as high magnitude antigen specific T-cells identified in the spleen. These data validate a genomic approach to antigen discovery and anti-tumor efficacy of Th1 selective vaccines for colon cancer prevention. Citation Format: Mary Disis, Ying Liu, Lauren Corulli, Erin Rodmaker, Denise Cecil. Vaccine approach for the interception of colon cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr IA016.