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.     

Antibody responses of Macaca fascicularis against a new inactivated polio vaccine derived from Sabin strains (sIPV) in DTaP-sIPV vaccine

Antibody responses of Macaca fascicularis against a new tetravalent vaccine composed of diphtheria toxoid, tetanus toxoid, acellular pertussis antigens, and inactivated poliovirus derived from Sabin strains (sIPV) was investigated to predict an optimal dose of sIPV in a new tetravalent vaccine (DTaP-sIPV) prior to conducting a dose-defined clinical study. Monkeys were inoculated with DTaP-sIPVs containing three different antigen units of sIPVs: Vaccine A (types 1:2:3 = 3:100:100 DU), Vaccine B (types 1:2:3 = 1.5:50:50 DU), and Vaccine C (types 1:2:3 = 0.75:25:25 DU). There was no difference in the average titers of neutralizing antibody against the attenuated or virulent polioviruses between Vaccines A and B. The average neutralizing antibody titers of Vaccine C tended to be lower than those of Vaccines A and B. The sIPV antigens did not affect the anti-diphtheria or anti-tetanus antibody titers of DTaP-sIPV. Furthermore, the average neutralizing antibody titers of Vaccine A against the attenuated and virulent polioviruses were comparable between M. fascicularis and humans. These results suggest that M. fascicularis may be a useful animal model for predicting the antibody responses to sIPVs in humans, and that it may be likely to reduce the amount of sIPVs contained in DTaP-sIPVs, even for humans.     

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

Background

The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine.

Methods

199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation.

Results

An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7⁺ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7⁺ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7⁺ IgA^- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus.

Discussion

Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.     

Visualizing the dynamic behavior of poliovirus plus-strand RNA in living host cells

Dynamic analysis of viral nucleic acids in host cells is important for understanding virus–host interaction. By labeling endogenous RNA with molecular beacon, we have realized the direct visualization of viral nucleic acids in living host cells and have studied the dynamic behavior of poliovirus plus-strand RNA. Poliovirus plus-strand RNA was observed to display different distribution patterns in living Vero cells at different post-infection time points. Real-time imaging suggested that the translocation of poliovirus plus-strand RNA is a characteristic rearrangement process requiring intact microtubule network of host cells. Confocal-FRAP measurements showed that 49.4 ± 3.2% of the poliovirus plus-strand RNA molecules diffused freely (with a D-value of 9.6 ± 1.6 × 10⁻¹⁰ cm²/s) within their distribution region, while the remaining (50.5 ± 2.9%) were almost immobile and moved very slowly only with change of the RNA distribution region. Under the electron microscope, it was found that virus-induced membrane rearrangement is microtubule-associated in poliovirus-infected Vero cells. These results reveal an entrapment and diffusion mechanism for the movement of poliovirus plus-strand RNA in living mammalian cells, and demonstrate that the mechanism is mainly associated with microtubules and virus-induced membrane structures.     

Persistence of Viruses in Desert Soils Amended with Anaerobically Digested Sewage Sludge

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log₁₀ reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 × 10⁵ Pa for both soils) and temperatures of 15, 27, and 40°C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40°C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40°C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15°C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log₁₀ reduction per day in clay loam and sandy loam soils, respectively. At 27°C, this rate increased to 0.629 log₁₀ reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15°C (k = 0.064 log₁₀ reduction per day, clay loam; k = 0.095 log₁₀ reduction per day, sandy loam) and 27°C (k = 0.133 log₁₀ reduction per day, clay loam; k = 0.154 log₁₀ reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40°C. No reduction of PRD-1 was observed after 28 days at 15°C and after 16 days at 27°C. At 40°C, the inactivation rates were 0.208 log₁₀ reduction per day in amended clay loam soil and 0.282 log₁₀ reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15°C, within 3 days at 27°C, and within 2 days at 40°C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.     

Cold chain and virus‐free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes

To prevent vaccine‐associated paralytic poliomyelitis, WHO recommended withdrawal of Oral Polio Vaccine (Serotype‐2) and a single dose of Inactivated Poliovirus Vaccine (IPV). IPV however is expensive, requires cold chain, injections and offers limited intestinal mucosal immunity, essential to prevent polio reinfection in countries with open sewer system. To date, there is no virus‐free and cold chain‐free polio vaccine capable of inducing robust mucosal immunity. We report here a novel low‐cost, cold chain/poliovirus‐free, booster vaccine using poliovirus capsid protein (VP1, conserved in all serotypes) fused with cholera non‐toxic B subunit (CTB) expressed in lettuce chloroplasts. PCR using unique primer sets confirmed site‐specific integration of CTB‐VP1 transgene cassettes. Absence of the native chloroplast genome in Southern blots confirmed homoplasmy. Codon optimization of the VP1 coding sequence enhanced its expression 9–15‐fold in chloroplasts. GM1‐ganglioside receptor‐binding ELISA confirmed pentamer assembly of CTB‐VP1 fusion protein, fulfilling a key requirement for oral antigen delivery through gut epithelium. Transmission Electron Microscope images and hydrodynamic radius analysis confirmed VP1‐VLPs of 22.3 nm size. Mice primed with IPV and boosted three times with lyophilized plant cells expressing CTB‐VP1co, formulated with plant‐derived oral adjuvants, enhanced VP1‐specific IgG1, VP1‐IgA titres and neutralization (80%–100% seropositivity of Sabin‐1, 2, 3). In contrast, IPV single dose resulted in <50% VP1‐IgG1 and negligible VP1‐IgA titres, poor neutralization and seropositivity (<20%, <40% Sabin 1,2). Mice orally boosted with CTB‐VP1co, without IPV priming, failed to produce any protective neutralizing antibody. Because global population is receiving IPV single dose, booster vaccine free of poliovirus or cold chain offers a timely low‐cost solution to eradicate polio.     

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.     

Environmental Poliovirus Surveillance during Oral Poliovirus Vaccine and Inactivated Poliovirus Vaccine Use in Córdoba Province,Argentina

This study compares the presence of environmental poliovirus in two Argentinean populations using oral poliovirus vaccine (OPV) or inactivated poliovirus vaccine (IPV). From January 2003 to December 2005, Córdoba City used IPV in routine infant immunizations, with the exception of intermittent OPV use in August 2005. Between May 2005 and April 2006, we collected weekly wastewater samples in Córdoba City and the province''s three major towns, which continued OPV use at all times. Wastewater samples were processed and analyzed for the presence of poliovirus according to WHO guidelines. During the months of IPV use in Córdoba City, the overall proportion of poliovirus-positive samples was 19%. During an intermittent switch from IPV to OPV, this proportion increased to 100% within 2 months. During the 3 months when IPV was reintroduced to replace OPV, a substantial proportion of samples (25%) remained positive for poliovirus. In the OPV-using sites, on average, 54% of samples were poliovirus positive. Seventy-seven percent of poliovirus isolates showed at least one mutation in the VP1-encoding sequence; the maximum genetic divergence from the Sabin strain was 0.7%. Several isolates showed mutations on attenuation markers in the VP1-encoding sequence. The frequency or type of virus mutation did not differ between periods of IPV and OPV use or by virus serotypes. This study indicates that the sustained transmission of OPV viruses was limited during IPV use in a middle-income country with a temperate climate. The continued importation of poliovirus and genetic instability of vaccine strains even in the absence of sustained circulation suggest that high poliovirus vaccine coverage has to be maintained for all countries until the risk of reintroduction of either wild or vaccine-derived poliovirus is close to zero worldwide.In the context of the near achievement of poliomyelitis eradication and anticipated cessation of oral poliovirus (PV) vaccine (OPV), the World Health Organization (WHO) has recommended the use of inactivated PV vaccine (IPV) in countries that have IPV production facilities or other countries where immunization programs fulfill certain financial and logistic criteria (37). IPV has been shown to be safe and immunogenic in children in both developed and developing countries.(34) IPV diminishes the excretion of PV by children challenged with the Sabin strain of PV only moderately. The questions of whether and to which extent Sabin PV that is reintroduced into a population immunized with IPV could establish circulation, mutate to vaccine-derived PV (VDPV), and consequently cause poliomyelitis remain important. No such emergence of VDPV in developed countries using IPV has been reported. However, suboptimal hygienic conditions and insufficient vaccine coverage in middle- or low-income countries could favor the establishment of PV circulation after reintroduction, as indicated by recent VDPV outbreaks in populations with low OPV coverage (27, 38).Argentina currently uses OPV in the childhood immunization program according to recommendations from the Pan-American Health Organization. The last case of poliomyelitis due to wild-type PV was reported in Argentina in 1984 and in Córdoba Province in 1971 (24). In Córdoba City, the capital of Córdoba Province, standalone IPV (Imovax Polio; Sanofi Pasteur) replaced OPV (Polioral; Novartis Vaccines) in the routine childhood immunization program (2, 4, and 6 months of age plus a booster at 18 months age) from 1 January 2003 to 31 December 2005, while the surrounding provinces continued to use OPV. Due to an IPV shortage between 10 August and 7 September 2005, OPV was used in the capital during this period. We conducted environmental PV surveillance in Córdoba Province from May 2005 to April 2006 to describe environmental PV circulation and molecular characteristics of PV depending on the vaccine used. In the present evaluation, we also describe the dynamic of PV circulation around the change of IPV-OPV-IPV-OPV in the capital. This observation can contribute evidence regarding the dynamics of PV circulation and its implication for global immunization policy after polio eradication.     

The maintaining of the active laboratory-based surveillance of the acute flaccid paralysis (AFP) cases in Romania in the framework of the strategic plan of the global polio eradication initiative

Until 2008 poliomyelitis was controlled in Romania by predominantly using Oral Poliovirus Vaccine Sabin (OPV); the alternative vaccination schedule (IPV formalin Inactivated Poliovirus Vaccine/OPV) will be implemented starting September 2008. The vaccination coverage with 4 doses of TOPV (trivalent oral polio vaccine) in the first 14 months of life has been > 90% since 1980. In Romania, the risk of the Vaccine-Associated Paralytic Poliomyelitis cases (VAPP) decreased from less than 2 VAPP cases/year in the 1995-2006 interval to 0 VAPP cases in 2007. The serological study was performed in 2006-2007 only in cases with pair serum samples from 28 acute flaccid paralysis (AFP) cases (age = 3 months - 14 years) and from 45 facial paralysis (FP) cases (age -6 months - 4 years 9 months). A high level of vaccinal coverage was shown for all poliovirus serotypes: >95% in AFP serum samples investigated; and for FP serum samples investigated the levels of antibodies against poliovirus (PV) serotypes were 98% for PV type 1; 87% for PV type 2: and 89% for PV type 3. If the European region is polio free since 2002, the risk of wild PV importation from endemic region remains present. The laboratory capacity for the fast detection and molecular investigations of the emergence of the new epidemic strains and a high level of population immunity must be maintained. A national seroprevalence study concerning all three PV serotypes must be performed.     

ON THE TEMPERATURE CHARACTERISTICS FOR FREQUENCY OF BREATHING MOVEMENTS IN INBRED STRAINS OF MICE AND IN THEIR HYBRID OFFSPRING. I

Young mice of a selected line of the dilute brown strain of mice exhibit over the range 15–25°C. (body temperature) a relation of frequency of breathing movements to temperature such that when fitted by the Arrhenius equation the data give a value for the constant µ of 24,000± calories or, less frequently, 28,000±. Young mice of an inbred albino strain show over the range 15–20°C. a value of µ = 34,000±, or, less frequently, 14,000±, with a critical temperature at about 20°C. and a value of µ = 14,000± above 20°C. The F₁ hybrids of these two strains, and the backcross generations to either parent strain, exhibit only those four values of the temperature characteristic observed in the parent strains and none other. One may therefore speak of the inheritance of the value of the constant µ, but the inheritance shows in this instance no Mendelian behavior. Furthermore there appears to be inherited the occurrence (or absence) of a critical temperature at 20°C. These experiments indicate the "biological reality" of the temperature characteristics.     

Isolation of Bacteria Which Can Grow at Both High pH and Low Temperature

Fifty bacterial strains able to grow at pH 10 and 0°C were isolated from soils, and growth characteristics of three selected strains were investigated. Strain 207, which showed the best growth rate of all the isolates at the conditions described above, could grow at a temperature of −5 to 39°C at pH 8.5. The optimum pH for this strain changed from 9.5 at 10°C to 9.0 at 20°C.     

Association of Virus with Cases of Rubella Studied in Toronto: Propagation of the Agent and Transmission to Monkeys

Using an interference test with indicator virus Echo 11, a virus has been isolated in nine of 18 specimens from cases of typical rubella. The virus will interfere with the development of cytopathology in green monkey kidney cells with viruses Echo 11, Coxsackie B1 and B4, Poliovirus I and III (Sabin strains) and simian virus SV4. In four of five paired sera this virus was neutralized by convalescent but not by the acute phase serum, tested by interference inhibition. No cytopathology was observed in unstained cultures or in sequential cultures stained with acridine orange or fluorescent antibody. The virus was destroyed by exposure to 56° C. for 30 minutes and 15% ether at 4° C. for 24 hours, but survived with some reduction in titre at 4° C. for 24 hours. Green monkeys infected by this virus developed a macular rash, lymphadenopathy and modest rise in white blood cell count.     

Capsid Functions of Inactivated Human Picornaviruses and Feline Calicivirus

The exceptional stability of enteric viruses probably resides in their capsids. The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined. Viruses were inactivated by UV, hypochlorite, high temperature (72°C), and physiological temperature (37°C), all of which are pertinent to transmission via food and water. Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States. The capsids of all 37°C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72°C. The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment. Inactivated HAV and FCV were captured by homologous antibodies. However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37°C-inactivated PV-1 was only partially captured. The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1. These findings indicate that the primary target of UV, hypochlorite, and 72°C inactivation is the capsid and that the target of thermal inactivation (37°C versus 72°C) is temperature dependent.     

Thermal Inactivation of Foot-and-Mouth Disease Viruses in Suspension

The heat resistance of foot-and-mouth disease virus (FMDV) strains isolated from outbreaks in Thailand was investigated in phosphate-buffered saline (PBS) at 50, 60, 70, 80, 90, and 100°C. The first-order kinetic model fitted most of the observed linear inactivation curves. The ranges of decimal-reduction time (D value) of FMDV strains at 50, 60, 70, 80, 90, and 100°C were 732 to 1,275 s, 16.37 to 42.00 s, 6.06 to 10.87 s, 2.84 to 5.99 s, 1.65 to 3.18 s, and 1.90 to 2.94 s, respectively. The heat resistances of FMDV strains at lower temperature (50°C) were not serotype specific. The effective inactivating temperature is approximately 60°C. Heat resistances of FMDV strains at 90 and 100°C were not statistically different (P > 0.05), while the FMDV serotype O (OPN) appeared to be the most heat resistant at 60 to 80°C. The other observed inactivation curves were linear with shoulder or tailing (biphasic curves). The shoulder effect was mostly observed at 90 and 100°C, while the tailing effect was mostly observed at 50 to 80°C. The adjusted D values in the case of shoulder and tailing effects did not affect the overall estimated heat resistance of these FMDV strains, so even unadjusted D values of deviant inactivation curves were legitimate. The z values of FMDV serotypes O, A, and Asia 1 were 21.78 to 23.26, 20.75 to 22.79, and 19.87°C, respectively. The z values of FMDV strains studied were not statistically significantly different (P > 0.05). The results of this study indicated that the heat resistance in PBS of FMDV strains from Thailand was much less than had been reported for foreign epidemic FMDV strains.     

Iron Superoxide Dismutase Protects against Chilling Damage in the Cyanobacterium Synechococcus species PCC7942

A strain of Synechococcus sp. PCC7942 lacking functional Fe superoxide dismutase (SOD), designated sodB⁻, was characterized by its growth rate, photosynthetic pigments, inhibition of photosynthetic electron transport activity, and total SOD activity at 0°C, 10°C, 17°C, and 27°C in moderate light. At 27°C, the sodB⁻ and wild-type strains had similar growth rates, chlorophyll and carotenoid contents, and cyclic photosynthetic electron transport activity. The sodB⁻ strain was more sensitive to chilling stress at 17°C than the wild type, indicating a role for FeSOD in protection against photooxidative damage during moderate chilling in light. However, both the wild-type and sodB⁻ strains exhibited similar chilling damage at 0°C and 10°C, indicating that the FeSOD does not provide protection against severe chilling stress in light. Total SOD activity was lower in the sodB⁻ strain than in the wild type at 17°C and 27°C. Total SOD activity decreased with decreasing temperature in both strains but more so in the wild type. Total SOD activity was equal in the two strains when assayed at 0°C.     

Rabbit as a Novel Animal Model for Hepatitis E Virus Infection and Vaccine Evaluation

Background

The identification of hepatitis E virus (HEV) from rabbits motivated us to assess the possibility of using rabbits as a non-human primate animal model for HEV infection and vaccine evaluation.

Methodology/Principal Findings

First, 75 rabbits were inoculated with seven strains of genotypes 1, 3, 4, and rabbit HEV, to determine the appropriate strain, administrative route and viral dosage. Second, 15 rabbits were randomly divided into three groups and vaccinated with 0 µg (placebo), 10 µg and 20 µg of HEV candidate vaccine, HEV p179, respectively. After three doses of the vaccination, the rabbits were challenged with 3.3×10⁵ genome equivalents of genotype 4 HEV strain H4-NJ703. The strain of genotype 1 HEV was not found to be infectious for rabbits. However, approximately 80% of the animals were infected by two rabbit HEV strains. All rabbits inoculated with a genotype 3 strain were seroconverted but did not show viremia or fecal viral shedding. Although two genotype 4 strains, H4-NJ153 and H4-NJ112, only resulted in part of rabbits infected, another strain of genotype 4, H4-NJ703, had an infection rate of 100% (five out of five) when administrated intravenously. However, only two out of fifteen rabbits showed virus excretion and seroconversion when inoculated orally with H4-NJ703 of three different dosages. In the vaccine evaluation study, rabbits vaccinated with 20 µg of the HEV p179 produced anti-HEV with titers of 1∶10⁴–1∶10⁵ and were completely protected from infection. Rabbits vaccinated with 10 µg produced anti-HEV with titers of 1∶10³–1∶10⁴ and were protected from hepatitis, but two out of the five rabbits showed virus shedding.

Conclusions/Significance

Rabbits may be served as an alternative to the non-human primate models for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.     

Modeling the Recovery of Heat-Treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 Spores at Suboptimal Temperature and pH Using Growth Limits

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.     

Functional Analysis of Antibodies against Dengue Virus Type 4 Reveals Strain-Dependent Epitope Exposure That Impacts Neutralization and Protection

Although prior studies have characterized the neutralizing activities of monoclonal antibodies (MAbs) against dengue virus (DENV) serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain- and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domain II (DII) and DIII of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after preincubation at 37°C. However, increasing the time of preincubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after preincubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4 and suggest that differences in epitope exposure relative to other DENV serotypes affect antibody neutralization and protective activity.     

Growth of Rhizobium japonicum Strains at Temperatures Above 27°C

A study was conducted to examine the growth responses of different Rhizobium japonicum strains to increasing temperatures, determine the degree of variability among strains in those responses, and identify temperature-related growth characteristics that could be used to select temperature-tolerant strains. Each of 42 strains was grown in liquid culture for 96 h at 19 incubation temperatures ranging from 27.4 to 54.1°C in a temperature gradient apparatus. Growth was estimated by measuring the change in optical density over time. Strains differed in their responses to increasing temperatures. Three characteristic temperatures were determined for each strain: the temperature giving the maximum optical density at 96 h (optimum temperature), the maximum temperature allowing a continuous increase in optical density during the 96-h period (maximum permissive temperature), and the maximum temperature allowing growth of the cultures after they were transferred to a uniform incubation temperature of 28°C (maximum survival temperature). The three characteristic temperatures varied among strains and had the following ranges: optimum temperature, from 27.4 to 35.2°C; maximum permissive temperature, from 29.8 to 38.0°C; and maximum survival temperature, from 33.7 to 48.7°C. Significant positive correlations were found between maximum permissive temperature and optimum temperature and between maximum permissive temperature and maximum survival temperature. Eight strains which had the highest maximum permissive temperature, optimum temperature, and maximum survival temperature were considered tolerant of high temperatures and were able to grow at temperatures higher than those previously reported for the most tolerant R. japonicum strains. The strains were of diverse geographical origin, but the response to high temperatures was not related to their origin. Evaluation of the temperature responses in pure culture may be useful in the search for R. japonicum strains better suited to environments in which high soil temperature is a limiting factor.