Immunization with the immediate-early tegument protein (open reading frame 62) of varicella-zoster virus protects guinea pigs against virus challenge.

The IE62 protein, the primary regulatory protein of varicella-zoster virus (VZV) and the major component of the virion tegument, was an effective immunogen in the guinea pig model of VZV infection, whereas the ORF 29 gene product, a nonstructural DNA replication protein, did not elicit protection. All animals immunized with the ORF 29 protein had cell-associated viremia compared with 2 of 11 guinea pigs given the IE62 protein (P = 0.005). VZV was detected in ganglia from 38% of the animals given the ORF 29 protein and 44% of the control animals compared with 9% of the animals immunized with the IE62 protein (P = 0.04). In contrast to the IE62 protein, immunization with the ORF 29 protein did not prime the animals for an enhanced T-cell response upon challenge with infectious virus. The VZV IE62 protein has potential value as a vaccine component.     

Effects of active immunization against gonadotropin releasing hormone on serum luteinizing hormone,progesterone levels and estrous cycles in the guinea pig

Mature female guinea pigs that had been observed to undergo three consecutive periods of estrus at approximately 16-day intervals were immunized with either 100 μg gonadotropin releasing hormone (GnRH) conjugated to 100 μg bovine serum albumin (BSA) or 100 μg BSA alone during diestrus (day 5–10) of the fourth cycle. Booster immunizations were administered 32 days after the first injection. Animals were bled by cardiac puncture at the time of first injection and at 16, 32, 48 and 64 days. Animals were necropsied at 64 days after first treatment.Daily observation indicated that vaginal manifestation of estrus was not apparent after a period equal to one estrous cycle in seven of ten GnRH immunized guinea pigs and after two cycles in the remaining three GnRH immunized guinea pigs. Estrous cycles persisted in BSA treated females throughout the experiment.Serum luteinizing hormone (LH) declined significantly by 32 days after the first immunization against GnRH and remained lower than both pretreatment values and levels in control animals at the same bleeding times throughout the experiment. Serum progesterone levels were significantly lower in the GnRH immunized group than in the control group at 48 and 64 days.At necropsy the weight of the ovaries of GnRH immunized guinea pigs was significantly lower than that of controls. Corpora lutea and antral follicles were present in both GnRH treated and control females. The presence of serum progesterone levels and of antral follicles in the GnRH immunized females suggests that a low level of gonadotropic support may have persisted to 64 days after initiation of treatment.Results indicate that immunization against GnRH can reduce LH and progesterone levels and induce cessation of estrous cycles in the guinea pig.     

Neutrophils Aid in Protection of the Vaginal Mucosae of Immune Mice against Challenge with Herpes Simplex Virus Type 2

Large numbers of polymorphonuclear leukocytes (PMNs) infiltrated the murine vaginal mucosa within 24 h after intravaginal inoculation with an attenuated strain of herpes simplex virus type 2 (HSV-2). The role of these cells in resolution of a primary genital infection and in protection of HSV-immune animals against challenge with a fully virulent HSV-2 strain was investigated. Depletion of greater than 95% of the PMNs at the vaginal mucosal surface prior to intravaginal inoculation with an attenuated HSV-2 strain resulted in significantly higher virus titers on days 3 to 7 but only slightly delayed resolution of the primary genital infection. These results suggest that neutrophils helped control the infection but that other immune mechanisms ultimately cleared the virus. Interestingly, depletion of PMNs from HSV-immune mice prior to challenge with a fully virulent HSV-2 strain resulted in a rise in virus titers to levels comparable to those of nonimmune mice and a more pronounced diminution of virus clearance from the vaginal mucosa despite the presence of HSV-specific B and T cells. Levels of gamma interferon (IFN-gamma) and HSV-specific antibody were comparable in neutrophil-depleted and control-treated immune mice following HSV-2 challenge, suggesting that RB6-8C5 treatment did not impair T- and B-cell function. Therefore, these results suggest that neutrophils play a role in limiting and clearing HSV-2 vaginal infections and that they are, in association with HSV-specific B and T cells, an important component in immune protection of the vaginal mucosa.     

Immunogenicity,Protective Efficacy,and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.     

Protection against experimental toxoplasmosis by adoptive immunotherapy

The role of humoral and cell-mediated immunity against toxoplasmosis in experimentally infected guinea pigs was examined by using a syngeneic passive transfer system. Serum or spleen and lymph node cells from guinea pigs immune to infection with the RH strain of Toxoplasma gondii conferred partial protection against symptomatic disease in recipient guinea pigs. This result was based on the reduced dissemination or growth of T. gondii parasites from the primary inoculation site to various selected organ sites of the recipients of immune serum or cells. Similar levels of partial protection against disseminated toxoplasmosis occurred in animals infused with cell suspensions enriched for immune T cells, whereas treatment of immune cells with a monoclonal anti-guinea pig T cell antibody plus complement abolished their ability to transfer resistance. These findings provide substantial direct evidence implicating both cellular and humoral components of the immune response as important effector mechanisms in host resistance to toxoplasmosis.     

Immune responses of inbred guinea pigs against random terpolymers containing L glutamic acid and L alanine.

The immune responses of the inbred guinea pig strains 2 and 13 have been determined against random terpolymers of L glutamic acid and L alanine and a third amino acid. Strain 2 guinea pigs responded against GAT10, GAT20(LLD), GAT10(NO2)15, GAT4, and GAL10. However, strain 13 guinea pigs responded only against GAT10. The explanation offered is that strain 2 guinea pigs, which have the Ir-GA gene, recognize the polymers via random GA determinants present in sufficient concentration in all of the above polymers. However, strain 13 guinea pigs recognize the GAT10 via the Ir-GT gene, and reduction in the concentration of tyrosyl residues below 10 mole % by various procedures alters the concentration of available random GT determinants necessary for interaction with the gene product of the Ir-GT gene.     

Genetically restricted immune responses in guinea pigs primed in vivo with antigen-bearing macrophages.

Guinea pigs injected intradermally with antigen pulsed macrophages generate a population of immune T cells that proliferate in vitro on second exposure to antigen. T cells from F1 (2 X 13) guinea pigs immunized with DNP-OVA on one parental macrophage respond in vitro only to DNP-OVA on macrophages identical to those used for immunization and not to DNP-OVA associated with the other parental macrophages. These results demonstrate that the immunogenicity of antigen is dependent upon the macrophages used for priming in that, with this approach, strain 2 or 13 guinea pigs immunized with allogeneic macrophages pulsed with antigen do not respond to either allogeneic or syngeneic antigen-bearing macrophages. However, lysates of antigen-pulsed macrophages can still immunize either allogeneic or syngeneic recipient via their own macrophages. F1 (2 X 13) guinea pigs are immunized by insulin B chain pulsed strain 13 macrophages (responder) but not by strain 2 macrophages (nonresponder) suggesting that whether a F1 (nonresponder X responder) guinea pig recognizes antigen bound to a parental macrophage is genetically restricted before immunization to the same extent as the donor parental macrophages used for immunization.     

Opsonizing activity of sera from animals immunized with pertussis vaccine

Opsonizing activity of guinea pig blood serum containing mercaptoethanol-resistant pertussis antibodies was studied in vitro on a model of microorganism ingestion by the mononuclears of the guinea pig peritoneal exudate. There were revealed distinct differences in the serum activity depending on the phagocytosis object. The blood serum of hyperimmunized rabbits stimulated the ingestion of Bordetella pertussis by mononuclears of guinea pigs--normal and immunized with pertussis vaccine. The blood sera of hyperimmunized guinea pigs and of mice immunized with pertussis vaccine twice displayed opsonins to B. pertussis. The blood sera of animals immunized with pertussis vaccine inhibited the staphylococcus ingestion by the peritoneal exudate mononuclears of guinea pigs, both normal and those immunized with pertussis vaccine.     

Guinea pig-adapted foot-and-mouth disease virus with altered receptor recognition can productively infect a natural host

We report that adaptation to infect the guinea pig did not modify the capacity of foot-and-mouth disease virus (FMDV) to kill suckling mice and to cause an acute and transmissible disease in the pig, an important natural host for this pathogen. Adaptive amino acid replacements (I(248)-->T in 2C, Q(44)-->R in 3A, and L(147)-->P in VP1), selected upon serial passages of a type C FMDV isolated from swine (biological clone C-S8c1) in the guinea pig, were maintained after virus multiplication in swine and suckling mice. However, the adaptive replacement L(147)-->P, next to the integrin-binding RGD motif at the GH loop in VP1, abolished growth of the virus in different established cell lines and modified its antigenicity. In contrast, primary bovine thyroid cell cultures could be productively infected by viruses with replacement L(147)-->P, and this infection was inhibited by antibodies to alphavbeta6 and by an FMDV-derived RGD-containing peptide, suggesting that integrin alphavbeta6 may be used as a receptor for these mutants in the animal (porcine, guinea pig, and suckling mice) host. Substitution T(248)-->N in 2C was not detectable in C-S8c1 but was present in a low proportion of the guinea pig-adapted virus. This substitution became rapidly dominant in the viral population after the reintroduction of the guinea pig-adapted virus into pigs. These observations illustrate how the appearance of minority variant viruses in an unnatural host can result in the dominance of these viruses on reinfection of the original host species.     

乙型脑炎减毒活疫苗(SA_(14)-14-2)在豚鼠体内保护作用的免疫机制的研究

本实验将乙脑减毒活疫苗ＳＡ_（１４）－１４－２株以不同疫苗病毒量（３．８７ＰＦＵ／ｍｌ和５．８７ＰＦＵ／ｍｌ）分别一次免疫豚鼠,观察其对强毒攻击后抑制毒血症和抗体形成的能力。结果显示疫苗（５．８７ＰＦＵ／ｍｌ）免疫组豚鼠攻击前虽然中和抗体阴性或很低,但经攻击感染后不同时间内均未出现病毒血症,对照组豚鼠则于第２,３,４天全部出现病毒血症。表明一次活疫苗免疫后能有效地抑制病毒血症的产生。免疫后３０天虽然免疫组的豚鼠中和抗体很低,但攻击感染后抗体迅速增长。第四天的抗体滴度为１：８～３２,第５天达１：１２８～２５６,第１４天抗体高达１：５１２～１０２４;而对照组抗体则上升很慢,第７天才出现低水平抗体（１：４）。血凝抑制抗体增长的动态与中和抗体近似。表明活疫苗免疫后虽然中和抗体水平不高,但一经感染可迅速产生高滴度抗体达到保护作用。     

Molecular cloning and expression of the IL-10 gene from guinea pigs

The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project is useful to directly clone much needed cDNAs necessary to study TB in the guinea pig. The newly cloned guinea pig IL-10 cDNA and recombinant proteins will serve as valuable resources for immunological studies in the guinea pig model of TB and other diseases.     

Immune correlates of protection against anthrax

Bacillus anthracis protective antigen (PA) has been produced from a recombinant B. subtilis and its efficacy, when combined with the Ribi adjuvant (MPL-TDW-CWS) or alhydrogel, has been compared with that of the licensed UK human vaccine, in guinea pigs challenged with aerosolized Ames strain spores. Recombinant PA combined with the Ribi adjuvant performed as well as PA from B. anthracis cultures in previous reports ( Ivins & Welkos 1986 ; Ivins et al . 1990 ; Turnbull et al . 1991 ; Jones et al . 1996 ; McBride et al . 1998 ) giving protection in 100% of animals exposed to the highest challenge dose of the Ames strain of B. anthracis that can be administered practically (retained lung doses of approximately 10⁶ spores).
In attempts at identifying markers of protection in immunized individuals, rPA in combination with the Ribi adjuvant induced a marker IgG₂ response in guinea pigs with no significant differences in IgG₁ levels when compared with other vaccine formulations ( McBride et al . 1998 ). In BALBc mice, rPA with the Ribi adjuvant induced a higher IgG_2a response compared with rPA with anhydrogel and the human vaccine.
To examine the role of anti-PA-specific antibodies in protection, guinea pig sera is being passively transferred into guinea pigs and SCID mice, followed by protection.
Similarly, B- and T-lymphocytes from immunized BALB/c mice are being separately and passively transferred into SCID mice with subsequent challenge. The neutralizing ability of the PA-specific antibodies is being studied using an in vitro macrophage lysis assay.     

Identification of the principal cell type yielding immune RNA capable of transferring tumor-specific cellular cytotoxicity

A search was made for the lymphoid cell type(s) which are the source of immune RNA (I-RNA) capable of transferring tumor-specific cell-mediated cytotoxicity (CMC). Hartley guinea pigs were immunized with syngeneic murine fibrosarcomas (BP-10 or BP-11) induced by 3,4-benzo(a)pyrene in C3H/HeJ mice, and the I-RNA was extracted individually from their spleens, lymph nodes, and peritoneal exudate (PE) cells. All three I-RNA preparations were able to convert normal C3H/HeJ mouse lymphocytes to effector cells significantly cytolytic to the specific syngeneic mouse tumor in vitro. Furthermore, lymphocytes and macrophages were purified from the spleens, lymph nodes, and PE cells of tumor-immunized guinea pigs. I-RNA was extracted from these purified cell populations and also from the pooled guinea pig lymphoid tissues. Normal C3H/HeJ lymphocytes were incubated with each type of I-RNA and tested in vitro for CMC against the specific tumor cells. Significant CMC against BP-10 targets was observed with mouse lymphocytes incubated with I-RNA extracted from pooled lymphoid tissues of BP-10 tumor-immunized guinea pigs. There was a reduced but still significant CMC when mouse lymphocytes were incubated with I-RNA extracted from purified guinea pig lymphocytes, whereas there was a markedly increased CMC when the I-RNA was extracted from purified guinea pig macrophages. As indicated by sucrose density gradient analysis, the lesser effectiveness of lymphocyte I-RNA was not due to RNA degradation resulting from lymphocyte purification or I-RNA extraction. Treatment of all types of I-RNA with RNase abrogated the transfer of CMC, whereas treatment of I-RNA with DNase or pronase did not. RNA extracted from the lymphoid tissues of guinea pigs immunized with complete Freund's adjuvant without tumor was ineffective. Mouse lymphocytes incubated with BP-10 macrophage I-RNA destroyed BP-10 but not BP-11 tumor cells, whereas lymphocytes incubated with BP-11 macrophage I-RNA killed BP-11 but not BP-10 tumor cells, thus indicating tumor specificity of the immunity transferred by macrophage I-RNA. Our results suggest that macrophages are the principal source of I-RNA capable of transferring tumor-specific CMC.     

Induction and Characterization of Neutralizing Antibodies against a Human Immunodeficiency Virus Type 1 Primary Isolate

Chimpanzees infected with the primary isolate DH012 mount potent neutralizing antibodies. This DH012 neutralizing activity is highly strain specific. Immune sera from guinea pigs immunized with recombinant DH012 gp120 could also neutralize this primary isolate. The neutralizing activity in chimpanzee and guinea pig sera against wild-type DH012 appears to be independent of a linear epitope in the V3 region of gp120. Interestingly, the neutralization escape mutant derived from growing DH012 in the presence of the potent neutralizing chimpanzee serum is at least 50-fold more sensitive than wild-type DH012 to neutralization by guinea pig immune sera. The unusually potent neutralizing activity against the DH012 neutralization-resistant virus is due to the presence of anti-V3 antibodies in guinea pig sera. These results suggested that recombinant gp120 could induce neutralizing antibodies against primary isolate DH012. The V3 of wild-type DH012 is poorly immunogenic in infected chimpanzees and is not accessible to neutralizing V3 antibodies. It is likely that this cryptic V3 region became exposed when the virus escaped the neutralizing activity of the chimpanzee serum.     

Complement-requiring neutralizing antibody in guinea pigs with primary and recurrent genital herpes

Guinea pigs inoculated intravaginally with herpes simplex virus type 2 (HSV-2) strain 1868 produced a serum complement-requiring neutralizing (CRN) antibody during primary acute infection, i.e., 10 days postinoculation. The CRN antibody titers in the guinea pig sera decreased to less than 1:10 after heating at 56 degrees C for 30 min. It was found that 32 units of complement were necessary to obtain a satisfactory HSV-2 neutralizing antibody titer. Nonheated sera significantly reduced virus infectivity titers when mixed with 3.5 log10 PFU of HSV-2 and incubated at 37 degrees C for 20 to 60 min (P less than 0.001), whereas the same sera after heating at 56 degrees C for 30 min showed no inhibitory effect. Only 27.3% of infected guinea pigs had low serum non-CRN antibody titers ranging from 1:20 to 1:40. In addition, no evidence of increase in CRN antibody titers was noted during spontaneous recurrent genital herpes infection.     

Long-term presence of virus-specific plasma cells in sensory ganglia and spinal cord following intravaginal inoculation of herpes simplex virus type 2

The tissue sites of long-term herpes simplex virus type 2 (HSV-2)-specific antibody production in mice and guinea pigs were identified. In addition to secondary lymphoid tissue and bone marrow, HSV-specific plasma cells were detected in spinal cords of mice up to 10 months after intravaginal inoculation with a thymidine kinase-deficient HSV-2 strain and in lumbosacral ganglia and spinal cords of guinea pigs inoculated with HSV-2 strain MS. The long-term retention of virus-specific plasma cells in the peripheral and central nervous systems following HSV infection may be important for resistance to reinfection of neuronal tissues or may play a role in modulation of reactivation from latency.     

Use of immunostimulatory sequence-containing oligonucleotides as topical therapy for genital herpes simplex virus type 2 infection

Synthetic oligonucleotides containing CpG motifs in specific sequence contexts have been shown to induce potent immune responses. We have evaluated mucosal administration of two immunostimulatory sequence (ISS)-containing phosphorothioate-stabilized oligonucleotides for antiherpetic efficacy in animal models. The ISS oligonucleotides, suspended in phosphate-buffered saline, were tested in mouse and guinea pig vaginal models of herpes simplex virus type 2 (HSV-2) infection. For comparison, groups of untreated, non-ISS oligonucleotide-treated, and acyclovir-treated animals also were monitored. The results indicated that vaginal epithelial application of ISS (up to 6 h after viral inoculation) with mice lethally challenged with HSV-2 delayed disease onset and reduced the number of animals that developed signs of disease (P = 0.003). ISS application significantly increased survival rates over those of controls (P = 0.0014). The ISS also impacted an established infection in the guinea pig model of HSV-2 disease. A single administration of ISS (21 days after viral inoculation) significantly reduced the frequency and severity of HSV-2 lesions compared to results with non-ISS oligonucleotide-treated and untreated guinea pigs (P < 0.01). HSV-2 is shed from the vaginal cavity of the guinea pig in the absence of lesions, similar to the case with humans. As an additional indication of ISS efficacy, the magnitude of viral shedding also was significantly reduced in ISS-treated animals (P < 0.001). These effects appeared to be immunologically mediated, since ISS had no direct effect on HSV-2 replication in vitro using standard plaque assays. These data suggest that ISS may be useful in the treatment and control of genital herpes in humans.     

Guinea pig chymase is leucine-specific: a novel example of functional plasticity in the chymase/granzyme family of serine peptidases

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the alpha clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig alpha-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by alpha1-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster alpha-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.     

Molecular cloning of a gene encoding a Chlamydia psittaci 57-kDa protein that shares antigenic determinants with ca. 60-kDa proteins present in many Gram-negative bacteria

In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.