Poliovirus Antigens Research Articles

Abstract B076: Novel use of Listeria and gemcitabine to improve immunotherapy for pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (pancreatic cancer), is the 4th leading cause of cancer deaths. This “silent killer” is characterized by its development of metastases often before the primary tumor can be detected, resulting in a five-year survival rate of only 4%. Gemcitabine and erlotinib, FDA-approved drugs for pancreatic cancer, improve median survival by less than six months in advanced stage patients, underscoring the need for additional and/or new alternative approaches. Cancer immunotherapy could be such new approach because of its great promise for metastatic cancer, however hurdles need to be overcome. The first hurdle is that tumor-associated antigens (TAA) used in most cancer vaccines are poorly immunogenic, and the second hurdle is immune suppression, particularly by myeloid-derived suppressor cells (MDSC) that prevent T cell activation in the tumor microenvironment (TME). To address these problems we have developed a novel strategy using an attenuated bacterium Listeria monocytogenes that delivers highly immunogenic antigens selectively into tumor cells of metastases and tumors combined with Gemcitabine (GEM) to reduce immune suppression through MDSC. We have cloned highly immunogenic recall antigens (RA) such as tetanus toxoid (TT), poliovirus (PV), and measlevirus (MV) antigens into the Listeria to activate existing memory T cells to foreign highly immunogenic antigens, which most individuals have been exposed to during childhood through vaccinations, and to deliver these antigens selectively into tumor cells. Memory T cells generated to the RA following childhood vaccinations circulate in their blood for life and can upon reactivation kill the infected tumor cells with high efficiency. We have previously demonstrated this with Listeria-TT in a triple-negative breast cancer model 4T1. Listeria and GEM also kill tumor cells through reactive oxygen species (ROS). In the current study we analyzed whether GEM could improve the efficacy of Listeria-TT immunotherapy in mice with pancreatic cancer (Panc-02 model). Experimental procedures: Tumor-bearing mice (pre-immunized with the human TT vaccine) were treated with GEM and Listeria-TT, and effects were analyzed on metastases, tumors, and MDCS. Results: We found that Listeria-TT combined with GEM resulted in a near complete elimination of metastatic pancreatic cancer (metastases and tumors), which was not possible with Listeria-TT or GEM alone. We also found that GEM reduced the MDSC population by 50% in the blood. Currently, we are analyzing CD8 T cell responses of treated and control mice with pancreatic cancer to immunodominant epitopes within the TT protein (TT1162-1169:SNWYFNHL and TT12-86-1294:LNIYYRRL) matching the H2-b haplotype. In conclusion, the combination of GEM and Listeria-TT eliminated metastatic pancreatic cancer in the Panc-02 model. We expect that this dramatic effect is a combination of immune-based and non-immune-based mechanisms. Future prospects: Since the cloned recall antigen fragments also contain many T cell epitopes recognized in humans, we expect that these constructs are directly translatable to human clinical trials in the near future. Finally, cancer patients could be tested in advance by revaccination with human childhood vaccines to determine to which antigens they react to best before starting the Listeria-recall antigen vaccine therapy. Based on patient's best response (most likely determined by MHC haplotype and the TME), a personalized therapy with the best combination of recall antigen(s) could be designed. Citation Format: Dinesh Chandra, Wilber Quispe-Tintaya, Arthee Jahangir, Alice Kwon, Rodrigo Alves da Silva, Lukman Solola, Ziqiang Yuan, Steven K. Libutti, Claudia Gravekamp. Novel use of Listeria and gemcitabine to improve immunotherapy for pancreatic cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B076.

Alternative inactivated poliovirus vaccines adjuvanted with Quillaja brasiliensis or Quil-a saponins are equally effective in inducing specific immune responses.

Inactivated polio vaccines (IPV) have an important role at the final stages of poliomyelitis eradication programs, reducing the risks associated with the use of attenuated polio vaccine (OPV). An affordable option to enhance vaccine immunogenicity and reduce costs of IPV may be the use of an effective and renewable adjuvant. In the present study, the adjuvant activity of aqueous extract (AE) and saponin fraction QB-90 from Quillaja brasiliensis using poliovirus antigen as model were analyzed and compared to a preparation adjuvanted with Quil-A, a well-known saponin-based commercial adjuvant. Experimental vaccines were prepared with viral antigen plus saline (control), Quil-A (50 µg), AE (400 µg) or QB-90 (50 µg). Sera from inoculated mice were collected at days 0, 28, 42 and 56 post-inoculation of the first dose of vaccine. Serum levels of specific IgG, IgG1 and IgG2a were significantly enhanced by AE, QB-90 and Quil-A compared to control group on day 56. The magnitude of enhancement was statistically equivalent for QB-90 and Quil-A. The cellular response was evaluated through DTH and analysis of IFN-γ and IL-2 mRNA levels using in vitro reestimulated splenocytes. Results indicated that AE and QB-90 were capable of stimulating the generation of Th1 cells against the administered antigen to the same extent as Quil-A. Mucosal immune response was enhanced by the vaccine adjuvanted with QB-90 as demonstrated by increases of specific IgA titers in bile, feces and vaginal washings, yielding comparable or higher titers than Quil-A. The results obtained indicate that saponins from Q. brasiliensis are potent adjuvants of specific cellular and humoral immune responses and represent a viable option to Quil-A.

Safety and Immunogenicity of a Toddler Dose Following an Infant Series of a Hexavalent Diphtheria, Tetanus, Acellular Pertussis, Inactivated Poliovirus, Haemophilus influenzae Type b, Hepatitis B Vaccine Administered Concurrently or at Separate Visits With a Heptavalent Pneumococcal Conjugate Vaccine

Combination diphtheria-tetanus-5 component acellular pertussis-inactivated poliovirus-Haemophilus influenzae b conjugate-hepatitis B vaccine (DTaP5-IPV-Hib-HepB) administered either concurrently with 7-valent pneumococcal conjugate vaccine (PCV7) or 1 month apart was generally safe and immunogenic at 2, 4 and 6 months of age. This study examined the effects of a booster dose at age 15 months. Participants were randomized to DTaP5-IPV-Hib-HepB plus PCV7, DTaP5-IPV-Hib-HepB with PCV7 administered 1 month later or a pentavalent DTaP5-IPV/Hib plus HepB plus PCV7 at 15 months of age in a randomized, open-label, phase IIb clinical trial. Immunogenicity endpoints were rates of seroresponse to pertussis toxoid, filamentous hemagglutinin, pertactin and fimbriae types 2 and 3; rates of seroprotection against (Hib) polyribosylribitol phosphate capsular polysaccharide, hepatitis B surface antigen, diphtheria toxoid, tetanus toxoid and poliovirus types 1, 2 and 3; and geometric mean titers to all vaccine antigens. Safety endpoints included solicited injection-site reactions and systemic and serious adverse events. Seroresponse/seroprotection rates for all antigens exceeded prespecified criteria in both groups that received the hexavalent DTaP5-IPV-Hib-HepB; in the group that received the currently licensed pentavalent vaccine, seroresponse/seroprotection rates exceeded the criteria for all antigens except filamentous hemagglutinin. Seroresponse rates were ≥88.9% for pertussis antigens and seroprotection rates against polyribosylribitol phosphate capsular polysaccharide, hepatitis B surface antigen, diphtheria toxoid, tetanus toxoid and poliovirus antigens were ≥95.1% in recipients of DTaP5-IPV-Hib-HepB. DTaP5-IPV-Hib-HepB administered concomitantly with PCV7 or 1 month apart at 15 months of age following the infant series was well-tolerated and elicited antibody responses to all vaccine antigens, with no significant interference from concomitant PCV7 administration (clinicaltrials.gov registration number NCT00362427).

Conformational Shift of a Major Poliovirus Antigen Confirmed by Immuno-Cryogenic Electron Microscopy

Small, interfacial conformational changes occur in some Ag-Ab interactions. Using cryogenic electron microscopy (cryo-EM), we have demonstrated such changes in a major antigenic site of a poliovirus capsid protein. During cell entry, native human poliovirus (160S particle) converts to a cell entry intermediate (135S particle) and later to an RNA-released (80S) particle. By mixing particles with Fabs of the neutralizing C3 mAb, we labeled the external loop connecting the B and C β-strands (BC loop) of the capsid protein VP1 (residues 95-105) in the 160S and 135S states. We then determined three-dimensional structures by cryo-EM and enhanced their interpretability by fitting high-resolution coordinates of C3 Fab and the capsid proteins into the density maps. Binding of C3 to either 160S or 135S particles caused residues of the BC loop, located on the tip of a prominent peak known as the "mesa," to move by an estimated 5 Å. C3 Abs are neutralizing and can bind bivalently. The orientation of the bound Fabs in our reconstructions suggests that C3 neutralizes poliovirus by binding two adjacent BC loops on the same mesa and inhibiting conformational changes in the viral capsid.

Safety and Immunogenicity of a Hexavalent Vaccine Administered at 2, 4 and 6 Months of Age With or Without a Heptavalent Pneumococcal Conjugate Vaccine

DTaP₅-IPV-Hib-HepB, an investigational hexavalent combination vaccine, was evaluated for safety and immunogenicity, when administered to infants with heptavalent pneumococcal conjugate vaccine (PCV7). Infants were randomized to receive DTaP₅-IPV-Hib-HepB plus PCV7, DTaP₅-IPV-Hib-HepB with PCV7 administered 1 month later or DTaP₅-IPV/Hib plus HepB plus PCV7 at 2, 4 and 6 months of age in an open-label, phase IIb trial. Vaccine responses were assessed by pertussis toxoid, filamentous hemagglutinin, pertactin and fimbriae types 2/3 seroconversion rates, Haemophilus influenzae type b polyribosylribitol phosphate capsular polysaccharide, hepatitis B surface antigen, diphtheria toxoid, tetanus toxoid and poliovirus types 1, 2 and 3 seroprotection rates and geometric mean titers. Solicited injection site and systemic reactions, serious adverse events, and other safety outcomes were reported. Seroprotection rates to polyribosylribitol phosphate, hepatitis B surface antigen, diphtheria toxoid, tetanus toxoid and poliovirus antigens across all groups met or exceeded predetermined acceptability criteria. Seroconversion rates to pertussis toxoid, pertactin and fimbriae types 2/3, but not filamentous hemagglutinin, met such criteria. Antidiphtheria antibodies were significantly lower when PCV7 was coadministered. Geometric mean titers to the other antigens of the hexavalent and PCV7 vaccines were all high and similar in the 2 groups. No safety signals were noted. DTaP₅-IPV-Hib-HepB administered at 2, 4 and 6 months of age concomitantly with PCV7 was well tolerated and elicited robust antibody responses to all but the antidiphtheria antigens for which there may be evidence of immune interference. Only filamentous hemagglutinin did not meet seroconversion rate acceptability criteria.

An open-label, randomized, multi-center study of the immunogenicity and safety of DTaP–IPV (Kinrix™) co-administered with MMR vaccine with or without varicella vaccine in healthy pre-school age children

In the US, it is recommended that 4-6 year old children receive diphtheria-tetanus-acellular pertussis (DTaP), inactivated poliovirus (IPV), measles-mumps-rubella (MMR), varicella (V), and influenza vaccines. Data relating to the concomitant administration of combination DTaP-IPV vaccine (Kinrix™; GlaxoSmithKline Biologicals) and influenza or V vaccines are currently limited. This study was undertaken to evaluate the immunogenicity and reactogenicity of Kinrix™ when co-administered with MMR (M-M-RII(®), Merck & Co.) and Varivax™ (Merck & Co.) in 4-6 year old children. Phase IIIb, open-label, non-inferiority study (NCT00871117). We randomized (1:1) healthy 4-6 year olds to receive Kinrix™+MMR+V on day 0 (Group 1), or Kinrix™+MMR on day 0, followed by V at month 1 (Group 2). We measured DTaP-IPV immunogenicity before and 1 month post-vaccination (prior to V vaccination in Group 2). We collected local and general solicited symptoms within 4 days after vaccination and serious adverse events (SAEs) through 6 months post-vaccination. We enrolled 478 subjects. One month post-vaccination, >95% of subjects in both groups had booster responses to diphtheria, tetanus and pertussis antigens and all subjects had seroprotective anti-poliovirus antibody titers. Immune responses in Group 1 were non-inferior to Group 2 for responses to DTaP-IPV antigens according to pre-specified criteria. Reporting of solicited local events at the DTaP-IPV site appeared to be similar between the two vaccine groups, as was reporting of solicited general adverse events within 4 days of vaccination; no vaccine related SAEs were reported. Concomitant administration of varicella vaccine with Kinrix™ and MMR did not impact the immunogenicity of diphtheria, tetanus, pertussis or poliovirus antigens. Both vaccine regimens were well tolerated. These results support the co-administration of DTaP-IPV, MMR, and V vaccines in 4-6-year-old children, providing protection against multiple diseases in a timely and efficient manner.

Immunogenicity of sabin inactivated poliovirus vaccine induced by diphtheria-tetanus-acellular pertussis and Sabin inactivated poliovirus combined vaccine

In order to search the preparation process and optimazing dosage ratio of adsorbed diphtheria-tetanus-acellular pertussis and sabin inactivated poliovirus combined vaccine (DTaP-sIPV), the neutralizing antibody titers of IPV induced by different concentration of DTaP-sIPV were investigated on rats. Two batches of DTaP-sLPV were produced using different concentration of sIPV and the quality control was carried. Together with sabin-IPV and DTaP-wIPV ( boostrix-polio, GSK, Belgium) as control group, the DTaP-sIPV were administrated on three-dose schedule at 0, 1, 2 month on rats. Serum sample were collected 30 days after each dose and neutralizing antibody titers against three types poliovirus were determined using micro-neutralization test. Two batches of prepared DTaP-sIPV and control sLPV were according to the requirement of Chinese Pharmacopoeia (Volume III, 2005 edition) and showed good stability. The seropositivity rates were 100% for sabin inactivated poliovirus antigen in all groups. The GMTs (Geometric mean titers) of neutralizing antibodies against three types poliovirus increased. The prepared DTaP-sIPV was safe, stable and effective and could induced high level neutralizing antibody against poliovirus on rats.

Immunogenicity and safety of combined adsorbed low-dose diphtheria, tetanus and inactivated poliovirus vaccine (REVAXIS®) versus combined diphtheria, tetanus and inactivated poliovirus vaccine (DT Polio®) given as a booster dose at 6 years of age

This randomized, comparative, phase-IIIb study conducted in France aimed to demonstrate whether seroprotection against diphtheria, tetanus and poliomyelitis 1 month after a single dose of REVAXIS (low-dose diphtheria) is non-inferior to seroprotection 1 month after a single dose of DT Polio (standard-dose diphtheria), both vaccines being given as a second booster to healthy children at 6 years of age. Children were randomly assigned to receive a single intramuscular dose of REVAXIS or DT Polio. Primary endpoints were the 1-month post-booster seroprotection rates for diphtheria, tetanus and poliovirus type-1, -2 and -3 antigens. Secondary endpoints were immunogenicity and safety observations. Of 788 children screened, 760 were randomized: REVAXIS group, 384 children; DT Polio group, 376 children. No relevant difference in demographic characteristics at baseline was observed between REVAXIS and DT Polio groups. Non-inferiority of REVAXIS compared with DT Polio for seroprotection was demonstrated against diphtheria (respectively 98.6% and 99.3%), tetanus (respectively 99.6% and 100%), and poliovirus antigens (100% for each types in both groups). No allergic reactions to REVAXIS were reported. A benefit/risk ratio in favor of REVAXIS was suggested by the trend towards a better tolerability of REVAXIS compared with DT Polio regarding the rate of severe solicited injection-site reactions. The results support the use of REVAXIS as a booster at 6 years of age in infants who previously received a three-dose primary series within the first 6 months of life and a first booster including diphtheria, tetanus and poliovirus vaccine(s) given before 2 years of age.

Immunogenicity and Reactogenicity of Diphtheria—Tetanus—Acellular Pertussis—Hepatitis B—Inactivated Poliovirus and Haemophilus influenzae Type B Vaccines Administered Concomitantly to Infants as a Three-dose Primary Course

Immunogenicity and reactogenicity of primary vaccination with combined diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated poliovirus (DTPa-HBV-IPV) vaccine when co-administered with Haemophilus influenzae (Hib) conjugate vaccine were assessed in 60 healthy infants. Infants received HBV vaccine at birth, then DTPa-HBV-IPV and Hib vaccines at age 1.5 months, 3.5 months and 6 months. Blood samples were collected before the first DTPa-HBV-IPV and Hib vaccine doses and 1 month after dose 3. Reactogenicity was assessed using diary cards. One month after primary vaccination, all infants were seroprotected/seropositive against all vaccine antigens evaluated. The poliovirus antigen could not be evaluated. The vaccines were well tolerated. No case of fever > 39.0 °C was reported. No serious adverse events were considered related to vaccination. Primary vaccination with DTPa-HBV-IPV and Hib vaccines was immunogenic and well tolerated. Combined vaccines, such as this pentavalent vaccine, minimize the number of injections and vaccination visits required to complete primary vaccination, and provide choice and flexibility for physicians and vaccine providers.

A simple quantitative affinity capturing assay of poliovirus antigens and subviral particles by single-domain antibodies using magnetic beads

Recently, single-domain recombinant antibody fragments (VHHs or nanobodies) against poliovirus type 1 were isolated. To examine the antigenicity of poliovirus using these recombinant VHHs, an alternative technique mimicking protein A immunoprecipitation had to be developed that was designed specifically for VHHs. The current study validated an affinity capturing assay that is based on the magnetic separation of unbound antigen and antigen–VHH complexes. The technique is simple, fast, reliable, quantitative and inexpensive and was employed to assess the reactivity of 15 VHHs for native infectious poliovirus (N antigen), heat-denatured virus (H antigen) and 14S subviral particles. Three distinct subsets of VHHs were tentatively distinguished based on their specificity for the antigens: one that binds only to 14S precursors, another that binds to the H antigen and a third that binds to the N antigen. Some VHHs of the latter two subsets bound 14S subviral particles with equal affinity but others had at least 100-fold less affinity for the precursors. All neutralizing VHHs were demonstrated to recognize N antigen and all N-specific VHHs were shown to be neutralizing. This study corroborates the findings that VHHs mainly target conformational epitopes and that they target additional epitopes as compared to classical antibodies. The described technique may be useful for epitope mapping and tracking conformational changes of proteins.

Co-administration of a novel Haemophilus influenzae type b and Neisseria meningitidis serogroups C and Y-tetanus toxoid conjugate vaccine does not interfere with the immune response to antigens contained in infant vaccines routinely used in the United States

An investigational combined Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroups C and Y tetanus toxoid conjugate vaccine (HibMenCY-TT) has been developed to protect infants from invasive disease caused by Hib and these meningococcal serogroups without adding injections to the immunization schedule. Incorporation of this novel vaccine into the US vaccination schedule will require demonstration of a lack of immunologic interference with other routine pediatric vaccines. This study assessed the immune response to 7-valent pneumococcal conjugate vaccine (PCV7) and combined diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated poliovirus vaccine (DTaP-HepB-IPV) when separately co-administered with HibMenCY-TT as compared to a US-licensed H. influenzae type b tetanus toxoid conjugate vaccine (Hib-TT) at 2, 4, 6 (N=606) and 12–15 months of age (N=366). HibMenCY-TT was non-inferior to Hib-TT in terms of antibody responses to all Streptococcus pneumoniae serotypes contained in PCV7 and the diphtheria, tetanus, pertussis, hepatitis B and poliovirus antigens contained in DTaP-HepB-IPV one month after the third vaccine dose, and the anti-tetanus geometric mean antibody concentration (GMC) was significantly higher in the HibMenCY-TT group than in the Hib-TT group. In an exploratory analysis, no significant differences in the proportion of subjects with anti-pneumococcal antibody concentrations ≥0.2 µg/ml or anti-pneumococcal GMC were seen between the two groups after the fourth vaccine dose. A schedule of HibMenCY-TT given concomitantly with PCV7 and DTaP-HepB-IPV would be expected to protect infants against all of the targeted diseases.

Study on defective interfering particles of Paramyxovirus, Tianjin strain enhanced the immune response of the mice

Objective To investigate whether the defective interfering(DI) particles of Paramyxovirus, Tianjin strain could be used as a candidate adjuvant. Methods DI particles were separated and purified. After being identified, it was equally mixed with the inactivated poliovirus (PV) vaccine. Kunming mice were administered subcutaneously with the mixture, besides we set groups of inactivated PV vaccine containing Al(OH) 3 as an adjuvant, inactivated PV vaccine of which the dose was doubled without any adjuvant, and negative control. Sera were collected in the 14th day after immunization, and the specific antibodies of PV were detected. T/B lymphocyte stimulation indexes(SI) were counted through the lymphocyte proliferation tests. The quantities of IFN-γ/IL-4 produced by the splenocytes which were stimulated again by PV antigen were detected. Results The SI and the quantity of IFN-γof the mice in the group of inactivated PV vaccine combining with DI particles of Paramyxovirus, Tianjin strain were more than other groups. Conclusion DI particles of Paramyxovirus, Tianjin strain could enhance the immune response of inactivated poliovirus, especially the cellullar immunologic response of Th1 type. Key words: Paramyxovirus, Tianjin strain; Defective interfering particles; Poliovirus; Immune response

Diphtheria-Tetanus-Acellular Pertussis and Inactivated Poliovirus Vaccines Given Separately or Combined for Booster Dosing at 4–6 Years of Age

In the United States, diphtheria-tetanus-acellular pertussis (DTaP) and inactivated poliovirus (IPV) booster vaccinations are recommended for children 4-6 years of age. A combined DTaP-IPV vaccine is being developed, which would reduce by one the number of injections in this age group. Children 4-6 years of age were randomized (1:1:1:1) to receive booster vaccination with 1 of 3 combined DTaP-IPV lots plus the measles, mumps, and rubella vaccine (N = 3156 for pooled lots) or separate doses of DTaP + IPV + measles, mumps, and rubella vaccine (N = 1053). Immunogenicity was assessed in a subset of children (N = 1331). Safety (solicited and unsolicited symptoms) including detailed assessment of local swelling reactions, was assessed in all children. Increases in antibody geometric mean concentrations/titers 1 month after vaccination were observed for the diphtheria, tetanus, acellular pertussis, and polio antigens. At least 92.2% of combined DTaP-IPV subjects and 92.6% of separate DTaP + IPV subjects had a postvaccination booster response for one or more DTaP antigens. Booster responses to one or more poliovirus antigens were observed in at least 96.6% of combined DTaP-IPV subjects and 92.8% of separate DTaP + IPV subjects. The combined DTaP-IPV vaccine was noninferior to separately administered DTaP and IPV vaccines with respect to DTaP antigen booster response rates and poliovirus antibody geometric mean titers ratios. Reporting of solicited local and systemic events was comparable between both groups. The combination DTaP-IPV vaccine provided immunogenicity and reactogenicity that is comparable to separately administered DTaP and IPV vaccines, with the advantage of requiring one less injection.

Development of an enzyme immunoassay for poliovirus antigens

An indirect solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Virus antigen was obtained in LLC-MK2 cell cultures and used to prepare antibodies in rabbit and guinea pig. Antibodies were evaluated by double immunodiffusion and neutralization test. Optimal concentrations of guinea pig and rabbit immunoglobulins were determined by checkerboard titration. Microtitre plates were coated with 15.0 µg/ml guinea pig anti-polio immunoglobulin and rabbit anti-polio immunoglobulin at the concentration of 7.94 µg/ml was used as detecting antibody. The standard curve with eight different antigen concentrations in eight replicates resulted in a coefficient of variation (CV) between 2.1% to 7.8%. The dose-response relationship was determined by simple linear regression with a coefficient of correlation (R²) equal to 96.4%. The assay detected a minimum of 2.3 µg/ml poliovirus antigen.

Virus-Specific CD4+and CD8+Cytotoxic T-Cell Responses and Long-Term T-Cell Memory in Individuals Vaccinated against Polio

The presence of poliovirus (PV)-specific CD4(+) T cells in individuals vaccinated against polio has been shown, but CD8(+) T-cell responses have not been described. Here, we functionally characterize the CD4(+) T-cell response and show for the first time that dendritic cells and macrophages can stimulate PV-specific CD8(+) T-cell responses in vitro from vaccinees. Both CD4(+) T and CD8(+) T cells secrete gamma interferon in response to PV antigens and are cytotoxic via the perforin/granzyme B-mediated pathway. Furthermore, the T cells also recognize and kill Sabin 1 vaccine-infected targets. The macrophage-stimulated CD4(+) T and CD8(+) T cells most likely represent memory T cells that persist for long periods in vaccinated individuals. Thus, immunity to PV vaccination involves not only an effective neutralizing antibody titer but also long-term CD4(+) and CD8(+) cytotoxic T-cell responses.

The Alpha/Beta Interferon Response Controls Tissue Tropism and Pathogenicity of Poliovirus

Poliovirus selectively replicates in neurons in the spinal cord and brainstem, although poliovirus receptor (PVR) expression is observed in both the target and nontarget tissues in humans and transgenic mice expressing human PVR (PVR-transgenic mice). We assessed the role of alpha/beta interferon (IFN) in determining tissue tropism by comparing the pathogenesis of the virulent Mahoney strain in PVR-transgenic mice and PVR-transgenic mice deficient in the alpha/beta IFN receptor gene (PVR-transgenic/Ifnar knockout mice). PVR-transgenic/Ifnar knockout mice showed increased susceptibility to poliovirus. After intravenous inoculation, severe lesions positive for the poliovirus antigen were detected in the liver, spleen, and pancreas in addition to the central nervous system. These results suggest that the alpha/beta IFN system plays an important role in determining tissue tropism by protecting nontarget tissues that are potentially susceptible to infection. We subsequently examined the expression of IFN and IFN-stimulated genes (ISGs) in the PVR-transgenic mice. In the nontarget tissues, ISGs were expressed even in the noninfected state, and the expression level increased soon after poliovirus infection. On the contrary, in the target tissues, ISG expression was low in the noninfected state and sufficient response after poliovirus infection was not observed. The results suggest that the unequal IFN response is one of the important determinants for the differential susceptibility of tissues to poliovirus. We consider that poliovirus replication was observed in the nontarget tissues of PVR-transgenic/Ifnar knockout mice because the IFN response was null in all tissues.

Analysis of antigenic profiles of inactivated poliovirus vaccine and vaccine-derived polioviruses by block-ELISA method

A new block-ELISA test for quantitative evaluation of relative reactivity of antigenic sites was developed and used to reveal the detailed epitope structure of inactivated poliovirus vaccines (IPV) and live poliovirus strains. Poliovirus was captured on ELISA plates coated with rabbit anti-poliovirus IgG and blocked by monoclonal antibodies (Mabs) specific to individual epitopes before the remaining reactive antigenic sites were quantified by polyclonal anti-poliovirus IgG conjugate. The decrease of conjugate binding by the pre-treatment with a Mab reflects its contribution to the overall reactivity of poliovirus antigen. The level of block activity of Mabs for a given antigen can be expressed as a percent of reduction of antigenic reactivity as determined by ELISA test. It can be normalized by expressing this value as a ratio to the block activity of a reference sample. The data on the blocking-activity of a panel of monoclonal antibodies specific to different antigenic sites represents the epitope composition (antigenic profile) of a sample. Quantitative differences in epitope composition were determined for nine samples of inactivated poliovirus vaccine (IPV) and compared with the International Reference Reagent. This method could be used for monitoring consistency of IPV production, comparison of vaccines made by different manufacturers, and for the analysis of antigenically modified strains of attenuated poliovirus. Antigenic structures of two isolates of type 1 vaccine-derived poliovirus (VDPV) were compared with the structures of parental Sabin 1 and wild-type Mahoney strains using 17 monoclonal antibodies and revealed significant differences, suggesting that the method can be used for screening of field isolates and rapid identification of antigenically divergent VDPV strains.

Gut-homing (alpha(4)beta(7)(+)) Th1 memory responses after inactivated poliovirus immunization in poliovirus orally pre-immunized donors.

Mucosal infections are prevented by a specialized local immune system. Immune cells of this compartment can also be found in the blood and are characterized by the expression of mucosa-specific homing molecules. Here, the cellular immune responses after inactivated poliovirus immunization (IPV) in poliovirus orally pre-immunized donors were investigated. Subcutaneous IPV induced a transient increase in the proliferative response against poliovirus antigen and in the number of poliovirus-specific CD4(+) T cells in the blood of the vaccinees. These cells were characterized to be of the effector memory type (CD45RA(-)/CD45RO(+)/CCR7(-)/CD27(+)) and expressed the homing molecule alpha(4)beta(7), indicating their origin from the gut. Together these data show the recurrence of gut-derived poliovirus-specific cells upon IPV and evaluate the whole-blood assay as a powerful tool for monitoring the success of a vaccination.

Pathogenesis of poliovirus infection in PVRTg mice: poliovirus replicates in peritoneal macrophages.

The pathogenesis of poliovirus infection, responsible for the induction of a poliovirus-specific mucosal immune response following intraperitoneal (i.p.) inoculation of virus in mice transgenic for the poliovirus receptor (PVRTg mice), was studied. Following inoculation of poliovirus, replication was determined by increase in virus titre (TCID(50)) and by PCR of poliovirus-specific negative-strand RNA in peritoneal macrophages, mesenteric lymph nodes, Peyer's patches, duodenum, brain, kidney and liver. The presence of poliovirus antigens in several cell types was detected by immunolabelling. It was demonstrated that poliovirus replicated in the peritoneal macrophages of PVRTg mice, since the virus titre in peritoneal cells was increased compared to the titre in the inoculum. Negative-strand RNA was detected in these cells and most of the poliovirus-immunostained cells had the morphology of macrophages and expressed the macrophage-specific markers CD86 and M1/70 on their surface. Furthermore, in peritoneal lavage, poliovirus was also present in CD19(+) B cells, but not in dendritic or T cells. Moreover, poliovirus was detected in macrophage-like cells in the lamina propria of the intestine, but not in epithelial cells. Replication of poliovirus in mesenteric lymph nodes, Peyer's patches and brain was followed by excretion of virus in the faeces. This suggests that the virus is transported due to migration of macrophages from the peritoneal cavity to mesenteric lymph nodes and the lamina propria of Peyer's patches. It is likely that this route is responsible for the induction of virus-specific IgA in the gut.

Combined hepatitis B vaccines

The status and likely impact of existing and potential new combined hepatitis B vaccines were broadly considered at the Viral Hepatitis Prevention Board (VHPB) meeting in Malta, October 2001. The currently available and/or licensed combined hepatitis B vaccines in Europe and the prospects for further such vaccines were reviewed. Data on the safety, immunogenicity, and European licensing status and availability of haxavalent vaccines combining hepatitis B (HepB), Haemophilus influenza type b (Hib), diphtheria, tetanus, and pertussis (acellular) (DTPa), and inactivated poliovirus (IPV) antigens were presented. Finally, the impact of the availability of combined hepatitis B vaccines on hepatitis B immunisation programmes in Europe were examined and the added value of combined hepatitis B vaccines globally was estimated.