Deletion of a decoy epitope in porcine circovirus 2 (PCV2) capsid protein affects the protective immune response in mice.

The swine pathogen porcine circovirus type 2 (PCV2) causes significant economic damage worldwide. The PCV2 capsid (CP) residues 169-STIDYFQPNNKR-180 have been identified as a decoy epitope that diverts the host immune response away from protective epitopes. However, the decoy epitope may include important linear or conformational protective epitopes against PCV2. In this study, we used the baculovirus system to express recombinant complete CP (1-233) and mutant CP (Δ169-180), in which the decoy epitope was deleted, and evaluated the immune response to these in mice. Immunization with mutant CP (Δ169-180) protein, which formed very low level of virus-like particles (VLPs), elicited significantly lower levels of PCV2 CP-specific IgG antibodies and a slightly lower neutralizing activity than immunization with the complete CP (1-233) protein. This finding suggests that the complete CP is important for efficient VLP assembly and induction of PCV2-specific IgG antibodies and neutralizing antibodies in mice. This study may provide useful information for next-generation vaccine design for PCV2 control.

Recombinant adenovirus carrying a core neutralizing epitope of porcine epidemic diarrhea virus and heat-labile enterotoxin B of Escherichia coli as a mucosal vaccine.

Porcine epidemic diarrhea virus (PEDV) targets the intestinal mucosa in pigs. To protect against PEDV invasion, a mucosal vaccine is utilized effectively. In this study, we generated a recombinant adenovirus vaccine encoding the heat-labile enterotoxin B (LTB) and the core neutralizing epitope (COE) of PEDV (rAd-LTB-COE). The fusion protein LTB-COE was successfully expressed by the recombinant adenovirus in HEK293 cells, and the immunogenicity of the vaccine candidate was assessed in BALB/c mice and piglets. Three intramuscular or oral vaccinations with rAd-LTB-COE at two-week intervals induced robust humoral and mucosal immune responses. Moreover, a cell-mediated immune response was promoted in immunized mice, and the neutralizing antibody inhibited both the vaccine strain and the emerging PEDV isolate. Immunization experiments in piglets revealed that rAd-LTB-COE was immunogenic and induced good immune responses in piglets. Further studies are required to evaluate the efficacy of rAd-LTB-COE against a highly virulent PEDV challenge.

Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus.

Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

Mutation of purD and purF genes further attenuates Brucella abortus strain RB51.

In the present study, transposon mutagenesis was used to further attenuate Brucella abortus RB51 vaccine strain. Two purD and purF mutants were constructed, characterized and evaluated for attenuation via intracellular survival in murine macrophage-like RAW264.7 and HeLa cells, and by clearance in BALB/c mice. The purD and purF mutants showed significantly decreased intracellular survival, and complementation of these mutants with intact copies of purD or purF genes of RB51 strain was able to restore these defects. In addition, the pur mutants presented significantly lowered persistence in mice. Immunization with purD and purF mutants protected mice against a challenge with the virulent B. abortus strain 544 at a level similar to that of the parent RB51. These data suggest that genes encoding the early stages of purine biosynthesis (purD and purF) are required for intracellular survival and virulence of B. abortus.

Branched-chain amino acid supplementation promotes aerobic growth of Salmonella Typhimurium under nitrosative stress conditions.

Nitric oxide (NO) inactivates iron-sulfur enzymes in bacterial amino acid biosynthetic pathways, causing amino acid auxotrophy. We demonstrate that exogenous supplementation with branched-chain amino acids (BCAA) can restore the NO resistance of hmp mutant Salmonella Typhimurium lacking principal NO-metabolizing enzyme flavohemoglobin, and of mutants further lacking iron-sulfur enzymes dihydroxy-acid dehydratase (IlvD) and isopropylmalate isomerase (LeuCD) that are essential for BCAA biosynthesis, in an oxygen-dependent manner. BCAA supplementation did not affect the NO consumption rate of S. Typhimurium, suggesting the BCAA-promoted NO resistance independent of NO metabolism. BCAA supplementation also induced intracellular survival of ilvD and leuCD mutants at wild-type levels inside RAW 264.7 macrophages that produce constant amounts of NO regardless of varied supplemental BCAA concentrations. Our results suggest that the NO-induced BCAA auxotrophy of Salmonella, due to inactivation of iron-sulfur enzymes for BCAA biosynthesis, could be rescued by bacterial taking up exogenous BCAA available in oxic environments.

Carbopol-incorporated thermoreversible gel for intranasal drug delivery.

The present study describes the preparation and evaluation of a poloxamer 407 (P407)-based thermoreversible gel using Carbopol 934P (C934P) as a mucoadhesive polymer and hydroxypropyl-ß-cyclodextrin (HP-ß-CD) for enhancing the aqueous solubility and intranasal absorption of fexofenadine hydrochloride (FXD HCl). The prepared gels were characterized by gelation temperature, viscoelasticity, and drug release profile. Thermoreversibility of P407/C934P gel was demonstrated by rheological studies. The incorporation of carbopol into P407 gel also reduced the amounts of drug released from the gel formulations (p < 0.05). In vivo pharmacokinetic results of the prepared gel formulations in rabbits (at 0.5 mg/kg dose) showed that the relative bioavailability of drug from P407/C934P gel was 11.3 and 2.7-fold higher than those of drug solution and P407 gel group, respectively. These findings suggested that developed thermoreversible gels could be used as promising dosage forms to improve intranasal drug absorption.

An immunoproteomic approach for characterization of the outer membrane proteins of Salmonella Gallinarum.

Salmonella enterica serovar Gallinarum (SG) is an important pathogen that causes fowl typhoid in chickens. In order to investigate SG outer membrane proteins (OMPs) as potential vaccine candidate proteins, we established a proteomic map and database of antigenic SG-OMPs. A total of 174 spots were detected by 2DE. Twenty-two antigen-reactive spots were identified as nine specific proteins using PMF. OmpA was the most abundant protein among all of the identified OMPs, and it exhibited seven protein species. We conducted Western blot analysis for the SG-OMPs in order to determine which proteins were cross-reactive to the serovars Salmonella Enteritidis, Salmonella Typhimurium, and SG. Our results indicated that OmpA was considered to be an antigenic cross-reactive protein among the three serovars. This study sheds new light on our understanding of cross-protection among Salmonella serovars.

A porcine circovirus type 2d-based virus-like particle vaccine induces humoral and cellular immune responses and effectively protects pigs against PCV2d challenge.

The pathogenic porcine circovirus type 2 (PCV2) leads to significant economic losses in pig production. PCV2d is currently the dominant genotype causing porcine circovirus-associated disease (PCVAD) worldwide. Therefore, development of a recombinant PCV2d-based vaccine is required to elicit complete protection against PCV2d infection. In this study, we generated virus-like particles of PCV2d-based capsid protein (Bac-2dCP) using a baculovirus expression system and evaluated its protective efficacy against PCV2d infection in specific pathogen-free (SPF) pigs. Three-week-old SPF miniature pigs were intramuscularly immunized with purified Bac-2dCP and intranasally challenged with PCV2d at 4 weeks post-vaccination. The Bac-2dCP group showed significantly higher IgG levels and neutralizing antibodies against PCV2b and PCV2d genotypes, as well as increased interferon-γ levels, and increased body weight and average daily weight gain compared with positive (challenged) and negative (unchallenged) controls. In particular, the Bac-2dCP group showed almost complete absence of PCV2d DNA in serum, nasal, and rectal swabs and in lung, lymph node, and kidney tissue samples. However, the positive control group exhibited low levels of neutralizing antibody, and high levels of PCV2 DNA in serum, swab, and tissue samples, resulting in PCV2-associated pathological lesions. The results of this study demonstrated that a recombinant Bac-2dCP vaccine conferred complete protection against a PCV2d challenge in SPF miniature pigs.

Characterization of Inflammasomes and Their Regulation in the Red Fox.

BACKGROUND: Inflammasomes recognize endogenous and exogenous danger signals, and subsequently induce the secretion of IL-1ß. Studying inflammasomes in the red fox (Vulpes vulpes) is crucial for wildlife veterinary medicine, as it can help control inflammatory diseases in foxes. METHODS: We investigated the activation and intracellular mechanisms of three inflammasomes (NLRP3, AIM2, and NLRC4) in fox peripheral blood mononuclear cells (PBMCs), using established triggers and inhibitors derived from humans and mice. RESULTS: Fox PBMCs exhibited normal activation and induction of IL-1ß secretion in response to representative inflammasome triggers (ATP and nigericin for NLRP3, dsDNA for AIM2, flagellin for NLRC4). Additionally, PBMCs showed normal IL-1ß secretion when inoculated with inflammasome-activating bacteria. In inhibitors of the inflammasome signaling pathway, fox inflammasome activation was compared with mouse inflammasomes. MCC950, a selective NLRP3 inhibitor, suppressed the secretion of dsDNA- and flagellin-mediated IL-1ß in foxes, unlike mice. CONCLUSIONS: These findings suggest that NLRP3 may have a common role in dsDNA- and flagellin-mediated inflammasome activation in the red fox. It implies that this fox inflammasome biology can be applied to the treatment of inflammasome-mediated diseases in the red fox.

Plant-expressed Zika virus envelope protein elicited protective immunity against the Zika virus in immunocompetent mice.

Zika virus infection causes multiple clinical issues, including Guillain-Barré syndrome and neonatal malformation. Vaccination is considered as the only strategy for the prevention of ZIKV-induced clinical issues. This study developed a plant-based recombinant vaccine that transiently expressed the ZIKV envelope protein (ZikaEnv:aghFc) in Nicotiana benthamiana and evaluated the protective immunity afforded by it in immunocompetent mice. ZikaEnv:aghFc induced both humoral and cellular immunity at a low dose (1-5 µg). This immune-inducing potential was enhanced further when adjuvanted CIA09A. In addition, antigen-specific antibodies and neutralizing antibodies were vertically transferred from immunized females to their progeny and afforded both protective immunity to ZIKV and cross-protection to Dengue virus infection. These results suggest that our plant-based ZIKV vaccine provides a safe and efficient protective strategy with a competitive edge.

Salmonella Typhimurium lacking phoBR as a live vaccine candidate against poultry infection.

Salmonella enterica serovar Typhimurium, with a broad-host range, is a predominant cause of non-typhoidal Salmonella infection in humans, and the infectious source is highly associated with food animals, especially poultry. Considering the horizontal transmission of S. Typhimurium from farm animals to humans, vaccination has been strongly recommended in industrial animals. In an effort to eradicate S. Typhimurium in poultry farms, a live candidate vaccine strain lacking the phoBR genes, which encode the PhoB/PhoR two-component regulatory system responsible for cellular phosphate signaling, was evaluated in mice and chickens. Lack of the phoBR genes promoted overgrowth of intracellular Salmonella. However, notably, in BALB/c mouse models, the ΔphoBR mutant showed attenuated virulence and instead, provided protection against infection with virulent Salmonella, thereby clearing out Salmonella in the spleen and liver. Accordingly, immunization with the ΔphoBR mutant increased immunoglobulin (Ig)G and IgM antibody responses and also tended to increase the IgG2a/IgG1 ratio, which is indicative of T helper (Th)1-mediated cellular immunity. In chicken challenge models, immunization with the ΔphoBR mutant significantly boosted the production of IgG and IgM antibodies after the second vaccination. The vaccinated chickens ceased fecal shedding of challenged Salmonella earlier than the non-vaccinated ones and showed no Salmonella in their caecum and ileum. These results demonstrate the potential of the S. Typhimurium ΔphoBR mutant as a vaccine in chickens.

Evaluation of Salmonella Typhimurium Lacking fruR, ssrAB, or hfq as a Prophylactic Vaccine against Salmonella Lethal Infection.

Non-typhoidal Salmonella (NTS) is one of the primary causes of foodborne gastroenteritis; occasionally, it causes invasive infection in humans. Because of its broad host range, covering diverse livestock species, foods of animal origin pose a critical threat of NTS contamination. However, there is currently no licensed vaccine against NTS infection. FruR, also known as Cra (catabolite repressor/activator), was initially identified as the transcriptional repressor of the fructose (fru) operon, and then found to activate or repress the transcription of many different genes associated with carbon and energy metabolism. In view of its role as a global regulator, we constructed a live attenuated vaccine candidate, ΔfruR, and evaluated its prophylactic effect against NTS infection in mice. A Salmonella Typhimurium mutant strain lacking fruR was defective in survival inside macrophages and exhibited attenuated virulence in infected mice. Immunization with the ΔfruR mutant stimulated the production of antibodies, including the IgG, IgM, and IgG subclasses, and afforded a protection of 100% to mice against the challenge of lethal infection with a virulent Salmonella strain. The prophylactic effect obtained after ΔfruR immunization was also validated by the absence of signs of hepatosplenomegaly, as these mice had comparable liver and spleen weights in comparison with healthy mice. These results suggest that the ΔfruR mutant strain can be further exploited as a promising vaccine candidate against Salmonella lethal infection.

Vaccination with a Zika virus envelope domain III protein induces neutralizing antibodies and partial protection against Asian genotype in immunocompetent mice.

BACKGROUND: Zika virus (ZIKV) is a mosquito-borne flavivirus classified in Flaviviridae family such as dengue (DENV), yellow fever, and West Nile virus. An outbreak of ZIKV infection can pose a major public health risk because the contagion is unpredictable and induces severe pathology such as Guillan-Barre syndrome and neonatal microcephaly. However, an authorized ZIKV vaccine is not yet available, while several vaccine candidates are under development. METHODS: In this study, we constructed a recombinant ZIKV vaccine (Z_EDIII) that includes ZIKV envelope protein domain III using E. coli expression system. Then both humoral and cellular immunity were examined in C57BL/6 (female, 8-weeks-old) mice via Indirect ELISA assay, PRNT, ELISpot and cytokine detection for IFN-γ, TNF-α, and IL-12. In addition, the cross protection against DENV was evaluated in pups from Z_EDIII vaccinated and infected dam. RESULTS: Mice immunized by Z_EDIII produced a significant amount of ZIKV EDIII-specific and neutralizing antibodies. Together with antibodies, effector cytokines, such as IFN-γ, TNF-α, and IL-12 were induced. Moreover, vaccinated females delivered the adaptive immunity to neonates who are protective against ZIKV and DENV challenge. CONCLUSIONS: This study observed Z-EDIII-induced humoral and cellular immunity that protected hosts from both ZIKV and DENV challenges. The result suggests that our ZIKV EDIII recombinant vaccine has potential to provide a new preventive strategy against ZIKV infection.

Zika virus baculovirus-expressed envelope protein elicited humoral and cellular immunity in immunocompetent mice.

Zika virus (ZIKV) is a mosquito-borne virus that has a high risk of inducing Guillain-Barré syndrome and microcephaly in newborns. Because vaccination is considered the most effective strategy against ZIKV infection, we designed a recombinant vaccine utilizing the baculovirus expression system with two strains of ZIKV envelope protein (MR766, Env_M; ZBRX6, Env_Z). Animals inoculated with Env_M and Env_Z produced ZIKV-specific antibodies and secreted effector cytokines such as interferon-γ, tumor necrosis factor-α, and interleukin-12. Moreover, the progeny of immunized females had detectable maternal antibodies that protected them against two ZIKV strains (MR766 and PRVABC59) and a Dengue virus strain. We propose that the baculovirus expression system ZIKV envelope protein recombinant provides a safe and effective vaccine strategy.

Evaluation of novel recombinant porcine circovirus type 2d (PCV2d) vaccine in pigs naturally infected with PCV2d.

INTRODUCTION: The pathogenic porcine circovirus type 2 (PCV2) causes significant economic losses in pig production. Emergence of the PCV2d genotype has been linked with PCV2-associated disease (PCVAD) outbreaks. However, no study has been conducted efficacy of an experimental PCV2d-based subunit vaccine in pigs. Therefore, PCV2b- and PCV2d-based capsid (CP) proteins were generated using a baculovirus (Bac) expression system, and we evaluated the protective immune responses in a commercial pig farm where predominant PCV2d is circulating. METHODS: Eighteen 3-week-old pigs with maternal antibodies were randomly divided into four groups, and were immunized with purified Bac-2dCP, mixed 1:1 ratio with purified Bac-2bCP and Bac-2dCP (Bac-mCP), a commercial PCV2a-based subunit vaccine (VAC) or phosphate-buffered saline (PBS) as controls. RESULTS: The Bac-2dCP and Bac-mCP groups had significantly higher PCV2b- or PCV2d- specific IgG and neutralizing antibody without interference by maternal antibody compared to control group in pigs naturally infected with PCV2d. Interestingly, not only serum IL-4 level was significantly increased in the Bac-2dCP group, but also PCV2d viremia level was significantly reduced than the control group. CONCLUSIONS: The recombinant Bac-2dCP subunit vaccine is a good candidate for the effective reduction against PCV2d infection.

Generation of a cold-adapted PRRSV with a nucleotide substitution in the ORF5 and numerous mutations in the hypervariable region of NSP2.

A cold-adapted porcine reproductive and respiratory syndrome virus (CA-VR2332) was generated from the modified live virus strain VR2332. CA-VR2332 showed impaired growth when cultured at 37°C with numerous mutations (S731F, E819D, G975E, and D1014N) in the hypervariable region of the NSP2, in which the mutation S731F might play a vital role in viral replication at 30°C. Conserved amino acid sequences of the GP5 protein suggests that CA-VR2332 is a promising candidate for producing an effective vaccine against PRRSV infection. Further studies on replication and immunogenicity in vivo are required to evaluate the properties of CA-VR2332.

Formation and Maturation of the Phagosome: A Key Mechanism in Innate Immunity against Intracellular Bacterial Infection.

Phagocytosis is an essential mechanism in innate immune defense, and in maintaining homeostasis to eliminate apoptotic cells or microbes, such as Mycobacterium tuberculosis, Salmonella enterica, Streptococcus pyogenes and Legionella pneumophila. After internalizing microbial pathogens via phagocytosis, phagosomes undergo a series of 'maturation' steps, to form an increasingly acidified compartment and subsequently fuse with the lysosome to develop into phagolysosomes and effectively eliminate the invading pathogens. Through this mechanism, phagocytes, including macrophages, neutrophils and dendritic cells, are involved in the processing of microbial pathogens and antigen presentation to T cells to initiate adaptive immune responses. Therefore, phagocytosis plays a role in the bridge between innate and adaptive immunity. However, intracellular bacteria have evolved diverse strategies to survive and replicate within hosts. In this review, we describe the sequential stages in the phagocytosis process. We also discuss the immune evasion strategies used by pathogens to regulate phagosome maturation during intracellular bacterial infection, and indicate that these might be used for the development of potential therapeutic strategies for infectious diseases.

Salmonella Typhimurium Lacking YjeK as a Candidate Live Attenuated Vaccine Against Invasive Salmonella Infection.

Non-typhoidal Salmonella (NTS) causes gastrointestinal infection, which is commonly self-limiting in healthy humans but may lead to invasive infection at extraintestinal sites, leading to bacteremia and focal systemic infections in the immunocompromised. However, a prophylactic vaccine against invasive NTS has not yet been developed. In this work, we explored the potential of a ΔyjeK mutant strain as a live attenuated vaccine against invasive NTS infection. YjeK in combination with YjeA is required for the post-translational modification of elongation factor P (EF-P), which is critical for bacterial protein synthesis. Therefore, malfunction of YjeK and YjeA-mediated EF-P activation might extensively influence protein expression during Salmonella infection. Salmonella lacking YjeK showed substantial alterations in bacterial motility, antibiotics resistance, and virulence. Interestingly, deletion of the yjeK gene increased the expression levels of Salmonella pathogenicity island (SPI)-1 genes but decreased the transcription levels of SPI-2 genes, thereby influencing bacterial invasion and survival abilities in contact with host cells. In a mouse model, the ΔyjeK mutant strain alleviated the levels of splenomegaly and bacterial burdens in the spleen and liver in comparison with the wild-type strain. However, mice immunized with the ΔyjeK mutant displayed increased Th1- and Th2-mediated immune responses at 28 days post-infection, promoting cytokines and antibodies production. Notably, the Th2-associated antibody response was highly induced by administration of the ΔyjeK mutant strain. Consequently, vaccination with the ΔyjeK mutant strain protected 100% of the mice against challenge with lethal invasive Salmonella and significantly relieved bacterial burdens in the organs. Collectively, these results suggest that the ΔyjeK mutant strain can be exploited as a promising live attenuated NTS vaccine.

Construction and immunization with double mutant ΔapxIBD Δpnp forms of Actinobacillus pleuropneumoniae serotypes 1 and 5.

Actinobacillus pleuropneumoniae (APP) causes a form of porcine pleuropneumonia that leads to significant economic losses in the swine industry worldwide. The apxIBD gene is responsible for the secretion of the ApxI and ApxII toxins and the pnp gene is responsible for the adaptation of bacteria to cold temperature and a virulence factor. The apxIBD and pnp genes were deleted successfully from APP serotype 1 and 5 by transconjugation and sucrose counter-selection. The APP1ΔapxIBDΔpnp and APP5ΔapxIBDΔpnp mutants lost hemolytic activity and could not secrete ApxI and ApxII toxins outside the bacteria because both mutants lost the ApxI- and ApxII-secreting proteins by deletion of the apxIBD gene. Besides, the growth of these mutants was defective at low temperatures resulting from the deletion of pnp. The APP1ΔapxIBDΔpnp and APP5ΔapxIBDΔpnp mutants were significantly attenuated compared with wild-type ones. However, mice vaccinated intraperitoneally with APP5ΔapxIBDΔpnp did not provide any protection when challenged with a 10-times 50% lethal dose of virulent homologous (APP5) and heterologous (APP1) bacterial strains, while mice vaccinated with APP1ΔapxIBDΔpnp offered 75% protection against a homologous challenge. The ΔapxIBDΔpnp mutants were significantly attenuated and gave different protection rate against homologous virulent wild-type APP challenging.