Chitosan-Deficient Cryptococcus as Whole-Cell Vaccines.

Creating a safe and effective vaccine against infection by the fungal pathogen Cryptococcus neoformans is an appealing option that complements the discovery of new small molecule antifungals. Recent animal studies have yielded promising results for a variety of vaccines that include live-attenuated and heat-killed whole-cell vaccines, as well as subunit vaccines formulated around recombinant proteins. Some of the recombinantly engineered cryptococcal mutants in the chitosan biosynthesis pathway are avirulent and very effective at conferring protective immunity. Mice vaccinated with these avirulent chitosan-deficient strains are protected from a lethal pulmonary infection with C. neoformans strain KN99. Heat-killed derivatives of the vaccination strains are likewise effective in a murine model of infection. The efficacy of these whole-cell vaccines, however, is dependent on a number of factors, including the inoculation dose, route of vaccination, frequency of vaccination, and the specific mouse strain used in the study. Here, we present detailed methods for identifying and optimizing various factors influencing vaccine potency and efficacy in various inbred mouse strains using a chitosan-deficient cda1Δcda2Δcda3Δ strain as a whole-cell vaccine candidate. This chapter describes the protocols for immunizing three different laboratory mouse strains with vaccination regimens that use intranasal, orotracheal, and subcutaneous vaccination routes after the animals were sedated using two different types of anesthesia.

A chemically induced attenuated strain of Candida albicans generates robust protective immune responses and prevents systemic candidiasis development.

Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a priority. Here, we could for the first time successfully attenuate the virulence of Candida albicans by treating it with a fungistatic dosage of EDTA and demonstrate it to be a potential live whole cell vaccine by using murine models of systemic candidiasis. EDTA inhibited the growth and biofilm formation of C. albicans. RNA-seq analyses of EDTA-treated cells (CAET) revealed that genes mostly involved in metal homeostasis and ribosome biogenesis were up- and down-regulated, respectively. Consequently, a bulky cell wall with elevated levels of mannan and ß-glucan, and reduced levels of total monosomes and polysomes were observed. CAET was eliminated faster than the untreated strain (Ca) as found by differential fungal burden in the vital organs of the mice. Higher monocytes, granulocytes, and platelet counts were detected in Ca- vs CAET-challenged mice. While hyper-inflammation and immunosuppression caused the killing of Ca-challenged mice, a critical balance of pro- and anti-inflammatory cytokines-mediated immune responses are the likely reasons for the protective immunity in CAET-infected mice.

Immunization with a whole cell vaccine reduces pneumococcal nasopharyngeal density and shedding, and middle ear infection in mice.

Pneumococcal Conjugate Vaccines (PCVs) have substantially reduced the burden of disease caused by Streptococcus pneumoniae (the pneumococcus). However, protection is limited to vaccine serotypes, and when administered to children who are colonized with pneumococci at the time of vaccination, immune responses to the vaccine are blunted. Here, we investigate the potential of a killed whole cell pneumococcal vaccine (WCV) to reduce existing pneumococcal carriage and mucosal disease when given therapeutically to infant mice colonized with pneumococci. We show that a single dose of WCV reduced pneumococcal carriage density in an antibody-dependent manner. Therapeutic vaccination induced robust immune responses to pneumococcal surface antigens CbpA, PspA (family 1) and PiaA. In a co-infection model of otitis media, a single dose of WCV reduced pneumococcal middle ear infection. Lastly, in a two-dose model, therapeutic administration of WCV reduced nasal shedding of pneumococci. Taken together, our data demonstrate that WCV administered in colonized mice reduced pneumococcal density in the nasopharynx and the middle ear, and decreased shedding. WCVs would be beneficial in low and middle-income settings where pneumococcal carriage in children is high.

A simple method for enrichment of phase I Coxiella burnetii.

Coxiella burnetii is the bacterial causative agent of the zoonosis Q fever. This bacterium undergoes lipopolysaccharide (LPS) phase transition similar to Enterobacteriaciae upon in vitro passage. Full-length, phase I C. burnetii LPS is a critical virulence factor and profoundly impacts vaccine-induced immunogenicity; thus, LPS phase is an important consideration in C. burnetii experimentation and Q fever vaccine design. Typically, phase I LPS-expressing organisms are obtained from the tissues of infected experimental animals. In this process, residual phase II LPS-expressing organisms are thought to be cleared by the host immune system. Here, we propose an efficient and non-animal-based method for the enrichment of C. burnetii phase I LPS-expressing bacteria in vitro. We utilize both Vero cell culture to selectively enrich solutions with phase I and intermediate phase LPS-expressing bacteria. This simple and quick method decreases reliance on experimental animals and is a sustainable solution for Q fever diagnostic and vaccine development hurdles.

Introduction of an Ultraviolet C-Irradiated 4T1 Murine Breast Cancer Whole-Cell Vaccine Model.

The advent of immunotherapy has revolutionized cancer treatments. However, the application of immune checkpoint inhibitors may entail severe side effects, with the risk of therapeutic resistance. The generation of chimeric antigen receptor (CAR) T-cells or CAR-NK cells requires specialized molecular laboratories, is costly, and is difficult to adapt to the rapidly growing number of cancer patients. To provide a simpler but effective immune therapy, a whole-cell tumor vaccine protocol was established based on ultraviolet C (UCV)-irradiated 4T1 triple-negative breast cancer cells. The apoptosis of tumor cells after UVC irradiation was verified using resazurin and Annexin V/propidium iodide flow cytometric assays. Protective immunity was achieved in immunized BALB/c mice, showing partial remission. Adoptive transfer of splenocytes or plasma from the mice in remission showed a protective effect in the naive BALB/c mice that received a living 4T1 tumor cell injection. 4T1-specific IgG antibodies were recorded in the plasma of the mice following immunization with the whole-cell vaccine. Interleukin-2 (IL-2) and oligonucleotide 2006 (ODN2006) adjuvants were used for the transfer of splenocytes from C57BL/6 mice into cyclophosphamide-treated BALB/c mice, resulting in prolonged survival, reduced tumor growth, and remission in 33% of the cases, without the development of the graft-versus-host disease. Our approach offers a simple, cost-effective whole-cell vaccine protocol that can be administered to immunocompetent healthy organisms. The plasma or the adoptive transfer of HLA-matching immunized donor-derived leukocytes could be used as an immune cell therapy for cancer patients.

T cell reactivity to Bordetella pertussis is highly diverse regardless of childhood vaccination.

The incidence of whooping cough due to Bordetella pertussis (BP) infections has increased recently. It is believed that the shift from whole-cell pertussis (wP) vaccines to acellular pertussis (aP) vaccines may be contributing to this rise. While T cells are key in controlling and preventing disease, nearly all knowledge relates to antigens in aP vaccines. A whole-genome mapping of human BP-specific CD4+ T cell responses was performed in healthy vaccinated adults and revealed unexpected broad reactivity to hundreds of antigens. The overall pattern and magnitude of T cell responses to aP and non-aP vaccine antigens are similar regardless of childhood vaccination, suggesting that asymptomatic infections drive the pattern of T cell reactivity in adults. Lastly, lack of Th1/Th2 polarization to non-aP vaccine antigens suggests these antigens have the potential to counteract aP vaccination Th2 bias. These findings enhance our insights into human T cell responses to BP and identify potential targets for next-generation pertussis vaccines.

Fructose potentiates the protective efficiency of live Edwardsiella tarda cell vaccine.

Vaccination is an effective measure to prevent infection by pathogens. Live vaccines have higher protective efficacy than inactivated vaccines. However, how live vaccines interact with the host from a metabolic perspective is unknown. The present study aimed to explore whether a live Edwardsiella tarda vaccine regulates host metabolism and whether this regulation is related to the protective efficacy of the vaccine. Therefore, a gas chromatography mass spectrometry (GC-MS)-based metabolomics approach was used to investigate the metabolomic profile of mice serum after vaccination with live E. tarda vaccine. Fructose was identified as a key biomarker that contributes to the immune protection induced by the live vaccine. Moreover, co-administration of exogenous fructose and the live vaccine synergistically promoted survival of mice and fish after bacterial challenge. These results indicate that metabolites, especially fructose, can potentiate the live E. tarda vaccine to increase its protective efficiency.

Using an Aluminum Hydroxide-Chitosan Matrix Increased the Vaccine Potential and Immune Response of Mice against Multi-Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, immobile, aerobic nosocomial opportunistic coccobacillus that causes pneumonia, septicemia, and urinary tract infections in immunosuppressed patients. There are no commercially available alternative antimicrobials, and multi-drug resistance is an urgent concern that requires emergency measures and new therapeutic strategies. This study evaluated a multi-drug-resistant A. baumannii whole-cell vaccine, inactivated and adsorbed on an aluminum hydroxide-chitosan (mAhC) matrix, in an A. baumannii sepsis model in immunosuppressed mice by cyclophosphamide (CY). CY-treated mice were divided into immunized, non-immunized, and adjuvant-inoculated groups. Three vaccine doses were given at 0D, 14D, and 28D, followed by a lethal dose of 4.0 × 108 CFU/mL of A. baumannii. Immunized CY-treated mice underwent a significant humoral response, with the highest IgG levels and a higher survival rate (85%); this differed from the non-immunized CY-treated mice, none of whom survived (p < 0.001), and from the adjuvant group, with 45% survival (p < 0.05). Histological data revealed the evident expansion of white spleen pulp from immunized CY-treated mice, whereas, in non-immunized and adjuvanted CY-treated mice, there was more significant organ tissue damage. Our results confirmed the proof-of-concept of the immune response and vaccine protection in a sepsis model in CY-treated mice, contributing to the advancement of new alternatives for protection against A. baumannii infections.

Non-antibiotic prevention and treatment against Acinetobacter baumannii infection: Are vaccines and adjuvants effective strategies?

Acinetobacter baumannii (A. baumannii) is a Gram-negative opportunistic pathogen widely attached to the surface of medical instruments, making it one of the most common pathogens of nosocomial infection, and often leading to cross-infection and co-infection. Due to the extensive antibiotic and pan-resistance, A. baumannii infection is facing fewer treatment options in the clinic. Therefore, the prevention and treatment of A. baumannii infection have become a tricky global problem. The requirement for research and development of the new strategy is urgent. Now, non-antibiotic treatment strategies are urgently needed. This review describes the research on A. baumannii vaccines and antibacterial adjuvants, discusses the advantages and disadvantages of different candidate vaccines tested in vitro and in vivo, especially subunit protein vaccines, and shows the antibacterial efficacy of adjuvant drugs in monotherapy.

Promising whole-cell vaccines against cryptococcosis.

Cryptococcosis is a mycosis caused by Cryptococcus neoformans and C. gattii species complexes. Although this infection is potentially lethal, no prophylactic vaccine is yet commercially available, and the immune memory that enables prevention is still under investigation. These pathogens have a capsule layer for immune evasion and a sophisticated mechanism to advance the infection, and it is expected that these characteristics will make it difficult to develop prophylactic vaccines and to decipher the protective immunity. The current vaccine studies are focused on subunit, mRNA, DNA, and viral vector vaccines, with whole-cell vaccines also proving successful against cryptococcal infections. Cryptococcal whole-cell vaccines have been composed of highly immunostimulating strains with low-pathogenicity that are modified by genetic recombination technology. Examples include the whole-cell vaccines H99γ, sgl1∆, fbp1∆, znf2oe , cda1/2/3∆, cap59∆, and cap60∆. Some of these whole-cell vaccines were found to be highly effective in prolonging life and suppressing the fungal burden after an infection challenge in mice, and to be cross-reactive to C. neoformans, C. gattii, and other fungal pathogens. Furthermore, for some vaccines, the protective effect can be retained even in an immunocompromised host depleted of CD4+ T cells. These findings have provided new insights into protective immunity that should aid in vaccine development. In this review, we highlight the upsides and downsides of whole-cell vaccines against cryptococcosis.

Standardization and Key Aspects of the Development of Whole Yeast Cell Vaccines.

In the context of vaccine development, improving antigenic presentation is critical for the activation of specific immune responses and the success of immunization, in addition to selecting an appropriate target. In this sense, different strategies have been developed and improved. Among them is the use of yeast cells as vehicles for the delivery of recombinant antigens. These vaccines, named whole yeast vaccines (WYVs), can induce humoral and cellular immune responses, with the additional advantage of dispensing with the use of adjuvants due to the immunostimulatory properties of their cell wall components. However, there are some gaps in the methodologies for obtaining and validating recombinant strains and vaccine formulations. The standardization of these parameters is an important factor for WYVs approval by regulatory agencies and, consequently, their licensing. This review aimed to provide an overview of the main parameters to consider when developing a yeast-based vaccine, addressing some available tools, and highlighting the main variables that can influence the vaccine production process.

Characterization of Coxiella burnetii Dugway Strain Host-Pathogen Interactions In Vivo.

Coxiella burnetii is a Gram-negative, intracellular bacterium that causes the zoonosis Q fever. Among the many natural isolates of C. burnetii recovered from various sources, the Dugway group exhibits unique genetic characteristics, including the largest C. burnetii genomes. These strains were isolated during 1954-1958 from wild rodents from the Utah, USA desert. Despite retaining phase I lipopolysaccharide and the type 4B secretion system, two critical virulence factors, avirulence has been reported in a guinea pig infection model. Using guinea pig models, we evaluated the virulence, whole-cell vaccine (WCV) efficacy, and post-vaccination hypersensitivity (PVH) potential of a representative Dugway strain. Consistent with prior reports, Dugway appeared to be highly attenuated compared to a virulent strain. Indeed, Dugway-infected animals showed similarly low levels of fever, body weight loss, and splenomegaly like Nine Mile II-infected animals. When compared to a human Q fever vaccine, QVax®, Dugway WCV exhibited analogous protection against a heterologous Nine Mile I challenge. PVH was investigated in a skin-testing model which revealed significantly decreased maximum erythema in Dugway Δdot/icm WCV-skin-tested animals compared to that of QVax®. These data provide insight into this unique bacterial strain and implicate its potential use as a mutated WCV candidate.

Vaccines against candidiasis: Status, challenges and emerging opportunity.

Candidiasis is a mycosis caused by opportunistic Candida species. The occurrence of fungal infections has considerably increased in the last few years primarily due to an increase in the number of immune-suppressed individuals. Alarming bloodstream infections due to Candida sp. are associated with a higher rate of morbidity and mortality, and are emerged as major healthcare concerns worldwide. Currently, chemotherapy is the sole available option for combating fungal diseases. Moreover, the emergence of resistance to these limited available anti-fungal drugs has further accentuated the concern and highlighted the need for early detection of fungal infections, identification of novel antifungal drug targets, and development of effective therapeutics and prophylactics. Thus, there is an increasing interest in developing safe and potent immune-based therapeutics to tackle fungal diseases. In this context, vaccine design and its development have a priority. Nonetheless, despite significant advances in immune and vaccine biology over time, a viable commercialized vaccine remains awaited against fungal infections. In this minireview, we enumerate various concerted efforts made till date towards the development of anti-Candida vaccines, an option with pan-fugal vaccine, vaccines in the clinical trial, challenges, and future opportunities.

A Nonadjuvanted Whole-Inactivated Pneumococcal Vaccine Induces Multiserotype Opsonophagocytic Responses Mediated by Noncapsule-Specific Antibodies.

Streptococcus pneumoniae (Spn) remains a major cause of global mortality, with extensive antigenic diversity between capsular serotypes that poses an ongoing challenge for vaccine development. Widespread use of pneumococcal conjugate vaccines (PCVs) targeting Spn capsules has greatly reduced infections by vaccine-included serotypes but has led to increased infections by nonincluded serotypes. To date, high cost of PCVs has also limited their usefulness in low-income regions where disease burdens are highest. To overcome these limitations, serotype-independent vaccines are being actively researched. We have developed a whole-cell gamma-irradiated Spn vaccine (termed Gamma-PN) providing serotype-independent protection. We demonstrate that Gamma-PN immunization of mice or rabbits via the clinically relevant intramuscular route induces protein-specific antibodies able to bind numerous nonvaccine encapsulated serotypes, which mediate opsonophagocytic killing and protection against lethal challenges. Gamma-PN induced comparable or superior opsonophagocytic killing assay (OPKA) responses in rabbits to the licensed Prevnar 13 vaccine (PCV13) for vaccine-included serotypes, and a superior response to nonincluded serotypes, including emergent 22F and 35B. Additionally, despite a lower observed reactogenicity, administration of Gamma-PN without adjuvant resulted in higher OPKA responses and improved protection compared to adjuvanted Gamma-PN. To our knowledge, this has not been demonstrated previously for a whole-inactivated Spn vaccine. Eliminating the requirement for adjuvant comes with numerous benefits for clinical applications of this vaccine and poses interesting questions for the inclusion of adjuvant in similar vaccines in development. IMPORTANCE The target pathogen of this study, Streptococcus pneumoniae, kills over 300,000 children <5 years of age every single year, and is the leading cause of pneumonia-associated mortality globally. While the capsular polysaccharide (CPS)-based vaccine Prevnar13 prevents serious illness caused by 13 serotypes, ongoing Prevnar13 use has driven the emergence of nonincluded serotypes as major causes of infection and disease. To overcome this issue, we have developed a next-generation pneumococcal vaccine conferring serotype-independent protection. This vaccine shows equivalent or superior efficacy to Prevnar13, and performance was heightened when our vaccine was administered with no adjuvant. These findings should be considered for similar vaccines in development, as the benefit of adjuvant is often assumed and its automatic inclusion may be limiting product efficacy, resulting in potential abandonment of viable vaccine candidates, or prolonging their time to clinic.

Current challenges in the manufacture of clinical-grade autologous whole cell vaccines for hematological malignancies.

Autologous whole cell vaccines use a patient's own tumor cells as a source of antigen to elicit an anti-tumor immune response in vivo. Recently, the authors conducted a systematic review of clinical trials employing these products in hematological cancers that showed a favorable safety profile and trend toward efficacy. However, it was noted that manufacturing challenges limit both the efficacy and clinical implementation of these vaccine products. In the current literature review, the authors sought to define the issues surrounding the manufacture of autologous whole cell products for hematological cancers. The authors describe key factors, including the acquisition, culture, cryopreservation and transduction of malignant cells, that require optimization for further advancement of the field. Furthermore, the authors provide a summary of pre-clinical work that informs how the identified challenges may be overcome. The authors also highlight areas in which future basic research would be of benefit to the field. The goal of this review is to provide a roadmap for investigators seeking to advance the field of autologous cell vaccines as it applies to hematological malignancies.

Leishmaniac Quest for Developing a Novel Vaccine Platform. Is a Roadmap for Its Advances Provided by the Mad Dash to Produce Vaccines for COVID-19?

"Bugs as drugs" in medicine encompasses the use of microbes to enhance the efficacy of vaccination, such as the delivery of vaccines by Leishmania-the protozoan etiological agent of leishmaniasis. This novel approach is appraised in light of the successful development of vaccines for Covid-19. All relevant aspects of this pandemic are summarized to provide the necessary framework in contrast to leishmaniasis. The presentation is in a side-by-side matching format with particular emphasis on vaccines. The comparative approach makes it possible to highlight the timeframe of the vaccine workflows condensed by the caveats of pandemic urgency and, at the same time, provides the background of Leishmania behind its use as a vaccine carrier. Previous studies in support of the latter are summarized as follows. Leishmaniasis confers life-long immunity on patients after cure, suggesting the effective vaccination is achievable with whole-cell Leishmania. A new strategy was developed to inactivate these cells in vitro, rendering them non-viable, hence non-disease causing, albeit retaining their immunogenicity and adjuvanticity. This was achieved by installing a dual suicidal mechanism in Leishmania for singlet oxygen (1O2)-initiated inactivation. In vitro cultured Leishmania were genetically engineered for cytosolic accumulation of UV-sensitive uroporphyrin I and further loaded endosomally with a red light-sensitive cationic phthalocyanine. Exposing these doubly dye-loaded Leishmania to light triggers intracellular production of highly reactive but extremely short-lived 1O2, resulting in their rapid and complete inactivation. Immunization of susceptible animals with such inactivated Leishmania elicited immunity to protect them against experimental leishmaniasis. Significantly, the inactivated Leishmania was shown to effectively deliver transgenically add-on ovalbumin (OVA) to antigen-presenting cells (APC), wherein OVA epitopes were processed appropriately for presentation with MHC molecules to activate epitope-specific CD8+ T cells. Application of this approach to deliver cancer vaccine candidates, e.g., enolase-1, was shown to suppress tumor development in mouse models. A similar approach is predicted to elicit lasting immunity against infectious diseases, including complementation of the spike protein-based vaccines in use for COVID-19. This pandemic is devastating, but brings to light the necessity of considering many facets of the disease in developing vaccination programs. Closer collaboration is essential among those in diverse disciplinary areas to provide the roadmap toward greater success in the future. Highlighted herein are several specific issues of vaccinology and new approaches worthy of consideration due to the pandemic.

Nanovaccines with cell-derived components for cancer immunotherapy.

Cancer nanovaccines as one of immunotherapeutic approaches are able to attack tumors by stimulating tumor-specific immunological responses. However, there still exist multiple challenges to be tackled for cancer nanovaccines to evoke potent antitumor immunity. Particularly, the administration of exogenous materials may cause the off-target immunotherapy responses. In recent years, biomimetic nanovaccines by using cell lysates, cell-derived nanovesicles, or extracted cell membranes as the functional components have received extensive attention. Such nanovaccines based on cell-derived components would show many unique advantages including inherent biocompatibility and the ability to trigger immune responses against a range of tumor-associated antigens. In this review article, we will introduce the recent research progresses of those cell-derived biomimetic nanovaccines for cancer immunotherapy, and discuss the perspectives and challenges associated with the future clinical translation of these emerging vaccine platforms.

JMM Profile: Bordetella pertussis and whooping cough (pertussis): still a significant cause of infant morbidity and mortality, but vaccine-preventable.

Whooping cough (pertussis) is a highly contagious respiratory bacterial infection caused by Bordetella pertussis and is an important cause of morbidity and mortality worldwide, particularly in infants. Bordetella parapertussis can cause a similar, but usually less severe pertussis-like disease. Bordetella pertussis has a number of virulence factors including adhesins and toxins which allow the organism to bind to ciliated epithelial cells in the upper respiratory tract and interfere with host clearance mechanisms. Typical symptoms of pertussis include paroxysmal cough with characteristic whoop and vomiting. Severe complications and deaths occur mostly in infants. Laboratory confirmation can be performed by isolation, detection of genomic DNA or specific antibodies. Childhood vaccination is safe, effective and remains the best control method available. Many countries have replaced whole-cell pertussis vaccines (wP) with acellular pertussis vaccines (aP). Waning protection following immunisation with aP is considered to be more rapid than that from wP. Deployed by resource-rich countries to date, maternal immunisation programmes have also demonstrated high efficacy in preventing hospitalisation and death in infants by passive immunisation through transplacental transfer of maternal antibodies.

Metal-Organic Framework Encapsulated Whole-Cell Vaccines Enhance Humoral Immunity against Bacterial Infection.

The increasing rate of resistance of bacterial infection against antibiotics requires next generation approaches to fight potential pandemic spread. The development of vaccines against pathogenic bacteria has been difficult owing, in part, to the genetic diversity of bacteria. Hence, there are many potential target antigens and little a priori knowledge of which antigen/s will elicit protective immunity. The painstaking process of selecting appropriate antigens could be avoided with whole-cell bacteria; however, whole-cell formulations typically fail to produce long-term and durable immune responses. These complications are one reason why no vaccine against any type of pathogenic E. coli has been successfully clinically translated. As a proof of principle, we demonstrate a method to enhance the immunogenicity of a model pathogenic E. coli strain by forming a slow releasing depot. The E. coli strain CFT073 was biomimetically mineralized within a metal-organic framework (MOF). This process encapsulates the bacteria within 30 min in water and at ambient temperatures. Vaccination with this formulation substantially enhances antibody production and results in significantly enhanced survival in a mouse model of bacteremia compared to standard inactivated formulations.

Fha Deficient Bordetella pertussis Isolates in Iran with 50 Years Whole Cell Pertussis Vaccination.

BACKGROUND: Bordetella pertussis, a highly contagious respiratory. Notably, the resurgence of pertussis has recently been associated with the lacking production of vaccine virulence factors. This study aimed to screen pertactin (Prn) and filamentous hemagglutinin (Fha) production in Iran with 50 years' whole cell vaccine (WCV) immunization program. METHODS: Overall, 130 B. pertussis isolates collected from Pertussis Reference Laboratory of Iran during 2005-2018. Real-time PCR was performed by targeting IS481, ptxP, IS1001 and IS1002 for species confirmation of B. pertussis. Western-blot was used to evaluate the expression of virulence factors (pertactin and filamentous hemagglutinin). RESULTS: All tested B. pertussis isolates expressed Prn and all except two isolates expressed Fha. We have sequenced genomes of these strains and identified differences compared with genome reference B. pertussis Tohama I. CONCLUSION: Many countries reporting Prn and Fha-deficiency due to acellular vaccine (ACV) pressure. Our results demonstrate in a country with WCV history, Fha-deficient isolates may rise independently. However, Prn-deficient isolates are more under the ACV pressure in B. pertussis isolates. Continues surveillance will provide a better understanding of the effect of WCV on the evolution of the pathogen deficiency.