Oral immunization against experimental salmonellosis I. Development of temperature-sensitive mutant vaccines

Mutant strains of Salmonella enteritidis were selected for their inability to proliferate at 37 C; when exposed to this temperature, these organisms formed tangled masses of long filaments in liquid media, presumably as a result of their inability to form cross septa. The mutants were also incapable of synthesizing flagella protein. A study of the biological charateristics of the mutants indicated that in most respects they resembled the parent strain of S. enteritidis; however, they were avirulent for mice, presumably because of the restriction of growth imposed by the body temperature of the animal. Preliminary studies have suggested that these mutants are highly effective in inducing protection against severe challenge infections of S. enteritidis; of especial interest is the fact that, when given orally, the mutants conferred a substantial degree of protection against oral infection with the virulent strain.     

Safety and efficacy of Mycoplasma gallisepticum TS-11 vaccine for the protection of layer pullets against challenge with virulent M. gallisepticum R-strain.

Mycoplasma gallisepticum TS-11 vaccine was studied for its safety and protective ability in 49-day-old M. gallisepticum-free and Mycoplasma synoviae-free commercial Tetra SL layer chickens. Sixty birds were distributed into four groups: 15 were unvaccinated but were challenged with M. gallisepticum R-strain, 15 were vaccinated by eye drop and then challenged with virulent M. gallisepticum R-strain 4 weeks post vaccination, 15 were designated as controls without vaccination and challenge, and 15 received TS-11 vaccine but no challenge. Based on the post-challenge clinical signs, body weight gain, gross pathological examination of air sacs and peritoneum, histological examination of the trachea, lung, spleen and liver, and reisolation of mycoplasmas from inner organs, the TS-11 vaccine is safe and does not produce clinical signs, a major decrease of body weight gain or pathological lesions. Vaccination induced a slight serological response to M. gallisepticum antigen in serum plate agglutination and blocking enzyme-linked immunosorbent assay tests and prevented development clinical signs of airsacculitis, peribronchitis and interstitial pneumonia on M. gallisepticum challenge.     

Pathogenicity for chickens of six strains of mycoplasma gallisepticum isolated from various birds

Pathogenicity of the type strain and five field strains of Mycoplasma gallisepticum isolated from various avian hosts was evaluated by chicken inoculation. Only two field strains isolated from chickens were highly pathogenic for the chicken respiratory tract.     

Characteristics of an influenza mutant temperature-sensitive for viral RNA synthesis

A ts mutant of fowl plague virus has been isolated after mutagenesis with 5-fluorouracil, which is temperature-sensitive in viral RNA synthesis. By temperature shift experiments it was demonstrated that the synthesis of viral particle RNA as well as complementary RNA is inhibited in vivo at the nonpermissive temperature (40 °), although the viral RNA polymerase could be detected by an in vitro test and was active in vitro at the nonpermissive temperature. When infected cells were incubated at the permissive temperature (33 °) for 3 hr and then shifted to 40 °, almost normal yields of viral proteins and activities including infectious particles were found.     

An SV40 mutant defective in VP4 expression exhibits a temperature-sensitive growth defect

On reexamination of temperature-sensitive D-type (tsD) mutants of simian virus 40 (SV40), we found that the tsD222 mutant is identical to the VP2 M228I mutant, which is defective in VP4 expression, at the nucleotide level. Although a previous study reported that lack of VP4 caused defects in viral dissemination in BSC-1 cells, this mutant showed a temperature-sensitive growth defect in CV-1 cells. tsD101:VP3 Q113K and tsD202:VP3 P108S exhibited a growth phenotype similar to that of tsD222, and they retained the VP4 open reading frame (ORF). These three mutants did not complement each other, suggesting that their defects were functionally indistinguishable. Transduction of the SV40 vector expressing wild-type VP4 in tsD222-infected cells did not ameliorate the growth defect at the non-permissive temperature. The results indicate that tsD mutation in minor capsid proteins has a more profound impact on viral propagation, and that lack of VP4 ORF seems to have little influence on viral growth.     

Characterization of a foot-and-mouth disease mutant temperature-sensitive for viral RNA synthesis

Summary A temperature-sensitive (ts) mutant of foot-and-mouth disease virus (FMDV) did not produce RNA polymerase activity nor synthesize viral RNA when incubated in cells solely at the nonpermissive temperature (38.5° C). Infected cells initially incubated at 38.5° C and then shifted down to 33° C synthesized increased amounts of viral RNA at earlier times compared to infected cells kept at 33° C throughout, indicating that RNA polymerase precursors were synthesized at 38.5° C. In cells shifted up to 38.5° C from 33° C, the total amount of viral RNA synthesized after infection increased sharply for about 15 minutes and then rapidly decreased over the next 2 hours. RNA polymerase activity presented a similar pattern in its initial twofold increase and subsequent rapid decrease. Pulse labeling experiments showed that mutant viral RNA synthesis continued at a diminishing rate for 2 hours in cells shifted up to 38.5° C. The data from temperature shift experiments indicated that essentially only the mutant RNA formed after shift-up was degraded. The FMDVts mutant is apparently additionally defective in being unable to protect viral RNA synthesized after shift-up to 38.5° C.With 5 FiguresMention of a trademark of proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

A temperature-sensitive mutant of Rous sarcoma virus that is defective for replication

ts 672 is a temperature sensitive mutant of the Prague strain of Rous sarcoma virus which fails to replicate infectious progeny virus at 41 °C. Chick embryo cells infected with ts 672 are transformed as evidenced by focus formation and increased rate of 2-deoxyglucose transport. Rescue of infectious progeny from ts 672-infected cells at 41 °C can be obtained by superinfection with avian leukosis viruses. Avian tumor virus group specific and type specific antigens can be detected in chick embryo cells infected with ts 672 at 41 °C, and physical particles that incorporate ³H-uridine and cosediment with avian tumor viruses on sucrose density gradients are produced in large numbers.     

Indirect ELISA for the detection of a specific antibody response against Mycoplasma gallisepticum

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for determining Mycoplasma gallisepticum antibodies in chicken sera. The M. gallisepticum antigen was detergent extracted and incorporated into ISCOMs. Sediment of broth medium treated with sarcosyl was used as control antigen. Sera were tested before and after absorption with broth medium components and ELJSA titres are expressed as optical density (OD) at 492 nm. Sera from experimentally or naturally infected chickens, those vaccinated with Salsbury Mg bacterin or both vaccinated and experimentally infected were compared with sera from M. gallisepticum free or SPF chickens. A high OD was observed when unabsorbed sera (even from SPF chickens) were tested with control antigen. The non-specific binding of M. gallisepticum negative sera could be removed by absorbing the sera with broth media components before the ELISA was performed. In contrast, ELISA titres obtained with sera from M. gallisepticum positive birds did not decrease significantly after absorption, except in the vaccinated and experimentally infected group. When the OD obtained with control antigen was subtracted from that obtained with ISCOM antigen, the mean value for M. gallisepticum free chickens was 0.083. Higher values were obtained with absorbed sera from experimentally or naturally infected (0.248-0.526), vaccinated (0.506), or vaccinated and infected (0.276-0.930) birds. The use of the ISCOM antigen presentation system in the indirect ELISA, combined with absorption of sera with broth components was demonstrated to be a useful diagnostic assay for M. gallisepticum antibodies.     

Protection of mice against virulent virus infection by a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary Experimental infection with HVJ (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) in mice was studied. Aerosol infection of newborn mice with the wild-type virus (HVJ-W) retarded the development of body weight and killed the animals within a few weeks. Large amounts of virus were isolated from both the lungs and the nasal turbinates of infected mice. In contrast, newborn mice exposed by inhalation to a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture showed no clinical signs and grew equally well as mock-infected animals. No infectious virus could be recovered from the lungs although thets mutant grew to moderate titre in the nasal turbinates.The prior inoculation of newborn mice with thets mutant virus induced a state of significant resistance to subsequent challenge with the virulent wild-type virus.No replication of challenge virus in both lungs and nasal turbinates could be detected and the animals were protected a lethal infection. It is suggested that an avirulent temperature-sensitive mutant which has lost the capacity to replicate in the lower respiratory tract but is still capable of multiplying in the nasal turbinates may be a promising candidate for use in live vaccines especially against the infectious disease of the lower respiratory tract.With 2 Figures     

A host-dependent temperature-sensitive mutant of Rous sarcoma virus: evidence for host factors affecting transformation

We have characterized a host range mutant of Rous sarcoma virus in order to identify host cell factors involved in transformation. This mutant, tsLA33-1, which was isolated from a stock of the temperature-sensitive mutant tsLA33, is not temperature-sensitive for transformation of chicken embryo fibroblasts, as judged by its ability to induce morphological changes and agar colony formation at both 36 and 41.5 degrees. In Rat-3 cells, however, this mutant induced a temperature-dependent transformation: infected Rat-3 cells were transformed at 34 degrees but not at 39.5 degrees. Retransformants were isolated from tsLA33-1-infected Rat-3 cells by growth in agar suspension at 39.5 degrees. Virus rescued from these retransformants induced a temperature-dependent transformation when reintroduced into rat cells. The level of expression of pp60v-src at 39.5 degrees was unchanged in the retransformants. When the retransformants were treated with herbimycin, an antibiotic which induces turnover of certain protein-tyrosine kinases, they reverted to a normal phenotype, indicating that the transformed phenotype of the retransformants was dependent on continued expression of pp60v-src. The retransformants are therefore pseudorevertants in which a cellular alteration has occurred that allows transformation at 39.5 degrees by the mutant pp60v-src. Thus the temperature-dependence of transformation by tsLA33-1 is affected by the cellular environment, and is suppressed or complemented both in chicken cells and in the rat cell pseudorevertants. No clear correlation between levels of phosphorylation at tyrosine and transformation was observed. In Rat-3 cells the pp60v-src encoded by tsLA33-1 may be defective in its interaction with low abundance substrates that are critical for transformation; alternatively the nonpermissive cells may require a higher threshold dose of pp60v-src for transformation.     

Genetic analysis of adenovirus type 2 VI. A temperature-sensitive mutant defective for DNA encapsidation

H2ts4 is a mutant defective in a late function. Centrifugation experiments in CsCl showed that at the nonpermissive temperature (39°) the mutant could produce only top components (TC). When examined in an electron microscope these TC appeared to be morphologically normal. Pulse-chase experiments revealed that proteins synthesized at 39° failed to assemble efficiently into virions when a shift-down to 33° was done immediately after the pulse. DNA encapsidation was completely inhibited when a chase was done at 39° prior to the shift-down to 33°. When [³⁵S]methionine-labeled polypeptides synthesized by infected cells were analyzed on sodium dodecyl sulfate-polyacrylamide gels it was found that at 39° ts4 was defective in the processing of PVII. The postcleavage polypeptides VII, VIII, X, XI, and XII were virtually absent. Temperature-shift experiments showed that processing was temperature sensitive in the shift-up experiments only. Electrophoresis of purified TC grown at 39° showed that the TC of ts4 differ from those of the wild type (wt) only in the slightly higher levels of polypeptides VIII, XI, and XII in the ts4-TC. Polypeptide X was absent from both wt and ts4-TC.     

Immunization with a single dose of a microencapsulated Brucella melitensis mutant enhances protection against wild-type challenge

The development of safe and efficacious immunization systems to prevent brucellosis is needed to overcome the disadvantages of the currently licensed vaccine strains that restrict their use in humans. Alginate microspheres coated with a protein of the parasite Fasciola hepatica (vitelline protein B [VpB]) and containing live Brucella melitensis attenuated mutant vjbR::Tn5 (BMEII1116) were evaluated for vaccine efficacy and immunogenicity in mice. A single immunization dose in BALB/c mice with the encapsulated vjbR mutant improved protection against wild-type B. melitensis 16M challenge compared to the nonencapsulated vaccine strain (P < 0.05). The encapsulated mutant was also shown to induce a sustained elevation of Immunoglobulin G levels. Cytokine secretion from spleen cells of mice vaccinated with the encapsulated vjbR::Tn5 revealed elevated secretion of gamma interferon and interleukin-12, but no interleukin-4, suggesting an induction of a T helper 1 response reflecting the enhanced immunity associated with microencapsulation. Together, these results suggest that microencapsulation of live attenuated organisms offers the ability to increase the efficacy of vaccine candidates.     

Induction of HIV-specific antibody response and protection against vaginal SHIV transmission by intranasal immunization with inactivated SHIV-capturing nanospheres in macaques

We have previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and that intranasal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody response in mice. In this study, to evaluate the protective effect of immunization, each three macaques was intranasally immunized with Con A-NS or inactivated simian/human immunodeficiency virus KU-2-capturing nanospheres (SHIV-NS) and then intravaginally challenged with a pathogenic virus, SHIV KU-2. After a series of six immunizations, vaginal anti-HIV-1 gp120 IgA and IgG antibodies were detected in all SHIV-NS-immunized macaques. After intravaginal challenge, one of the three macaques in each of the Con A-NS- and SHIV-NS-immunized groups was infected. Plasma viral RNA load of infected macaque in SHIV-NS-immunized macaques was substantially less than that in unimmunized control macaque and reached below the detectable level. However, it could not be determined whether intranasal immunization with SHIV-NS is effective in giving complete protection against intravaginal challenge. To explore the effect of the SHIV-NS vaccine, the remaining non-infected macaques were rechallenged intravenously with SHIV KU-2. After intravenous challenge, all macaques became infected. However, SHIV-NS-immunized macaques had lower viral RNA loads and higher CD4(+) T cell counts than unimmunized control macaques. Plasma anti-HIV-1 gp120 IgA and IgG antibodies were induced more rapidly in the SHIV-NS-immunized macaques than in the controls. The rapid antibody responses having neutralizing activity might contribute to the clearance of the challenge virus. Thus, SHIV-NS-immunized macaques exhibited partial protection to vaginal and systemic challenges with SHIV KU-2.     

Immediate protection of mice from lethal wild-type Sendai virus (HVJ) infections by a temperature-sensitive mutant, HVJpi, possessing homologous interfering capacity

Protection of mice from lethal Sendai virus (HVJ) infections by a temperature-sensitive mutant, HVJpi, which was isolated from a carrier culture, was studied. HVJpi had a strong interfering capacity with the replication of virulent wild-type virus in LLCMK2 cells. When a high dose of HVJpi (3.0 x 10(7) CIU) was inoculated intranasally into mice, the mice showed neither illness nor lung lesions but gained significant resistance against the challenge of virulent wild-type virus (18 LD50) immediately after inoculation. In contrast, the mice inoculated with a lower dose of HVJpi (8.2 x 10(5) CIU) did not show the immediate resistance but became immune several days after inoculation. Time courses of the virus replication in the lung revealed that the replication of wild-type virus was strongly suppressed to about 1/1000 by the simultaneous infection with a high dose of HVJpi, thus resulting in minimizing the lung lesions and survival of all the mice infected. Neither interferon nor natural killer cells appeared to play a major role in the immediate immune status by HVJpi, since no difference was observed in protection of mice simultaneously infected with wild-type virus and HVJpi in spite of pretreatment of the mice with anti-interferon and anti-asialo GM1 antibodies as compared with that of the untreated doubly infected mice. On the other hand, it was suggested by analysis of viral polypeptides synthesized in the lung of infected mice by Western blotting that the early stage of replication of wild-type virus in the lung was inhibited mainly by the interfering capacity of HVJpi. These results indicate that HVJpi is an unique virus mutant which is capable of protecting mice from lethal Sendai virus infections by its interfering capacity immediately after inoculation and then by the induction of virus-specific immune responses.     

An evolutionarily-conserved role for murine Ly-1 B cells in protection against bacterial infections.

The murine Ly-1 B cell lineage, although comprising only a minority of peripheral IgM+ B cells, secretes a major proportion of the IgM antibodies occurring naturally in serum. Ly-1 B cells also seed a large number of IgA+ plasma cells to the gut walls, thereby contributing significantly to production of natural IgA antibodies in response to chronic stimulation by the normal gut flora. Apart from these naturally-produced antibodies, Ly-1 B cells also produce specific antibodies following deliberate immunisation with the bacterial cell wall antigens, phosphorylcholine and dextran. The inability of the X-linked immunodeficient CBA/N mice to produce antibody responses to these two antigens is overcome by reconstitution with normal Ly-1 B cells from the parental CBA strain. Ly-1 B cells therefore appear to play a dominant role in natural immunity and protection against bacterial infections. The compartmentalisation of development and function within murine B cells is suggestive of an evolutionary structuring of the murine immune system, with Ly-1 B cells representing a conserved, primitive B cell lineage and retaining key, associated functions.     

Selection for temperature-sensitive mutations in specific vaccinia virus genes: isolation and characterization of a virus mutant which encodes a phosphonoacetic acid-resistant, temperature-sensitive DNA polymerase

Seven temperature-sensitive mutants of vaccinia virus have been isolated after preselection for virus resistant to phosphonoacetic acid (PAA). In all seven mutants, the PAA-resistant (PAAr) and ts lesions represent separate mutations. In one mutant, NG26, the PAAr (NG26-PAAr) and ts (NG26-ts) mutations are very closely linked. Both NG26-ts and NG26-PAAr map in the HindIII E DNA fragment. NG26 has a DNA-negative phenotype at 40 degrees. NG26-ts is in the same complementation group as ts42, another DNA-negative mutant which maps in the HindIII E DNA fragment (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128, 000-000, 1983). The order of the mutations is (NG26-ts)-(NG26-PAAr)-ts42. The virus-coded DNA polymerase has been partially purified from wt- and NG26-infected cells. The DNA polymerase encoded by NG26 is temperature sensitive and PAA resistant in vitro as compared to the wt enzyme.