Use of Vaxfectin adjuvant with DNA vaccine encoding the measles virus hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques against measles virus

A measles virus vaccine for infants under 6 months of age would help control measles. DNA vaccines hold promise, but none has provided full protection from challenge. Codon-optimized plasmid DNAs encoding the measles virus hemagglutinin and fusion glycoproteins were formulated with the cationic lipid-based adjuvant Vaxfectin. In mice, antibody and gamma interferon (IFN-γ) production were increased by two- to threefold. In macaques, juveniles vaccinated at 0 and 28 days with 500 μg of DNA intradermally or with 1 mg intramuscularly developed sustained neutralizing antibody and H- and F-specific IFN-γ responses. Infant monkeys developed sustained neutralizing antibody and T cells secreting IFN-γ and interleukin-4. Twelve to 15 months after vaccination, vaccinated monkeys were protected from an intratracheal challenge: viremia was undetectable by cocultivation and rashes did not appear, while two naïve monkeys developed viremia and rashes. The use of Vaxfectin-formulated DNA is a promising approach to the development of a measles vaccine for young infants.     

Discrepancy in Dengue Virus Neutralizing Antibody Titers between Plaque Reduction Neutralizing Tests with Fcγ Receptor (FcγR)-Negative and FcγR-Expressing BHK-21 Cells

Protective immunity against dengue virus (DENV) is best reflected by the presence of neutralizing antibodies. The conventional plaque reduction neutralizing test (PRNT) is performed using Fcγ receptor (FcγR)-negative cells. Because FcγR plays a key role in antibody-dependent enhancement, we examined neutralizing antibody titers of mouse monoclonal antibodies and human serum samples in PRNTs using FcγRIIA-negative and FcγRIIA-expressing BHK cells. There was a discrepancy in dengue virus neutralizing antibody titers between PRNTs using FcγRIIA-negative versus FcγRIIA-expressing BHK cells. Neutralizing antibody titers to DENV-1 and DENV-2 tested with monoclonal antibodies, and with most of the human serum samples, were higher in assays using BHK cells than those using FcγRIIA-expressing BHK cells. The results suggest that neutralizing antibody titers determined using FcγRIIA-expressing cells may better reflect the protective capacity of anti-DENV antibodies, as the major target cells of DENV infection are FcγR-positive cells.Dengue virus (DENV), a member of the family Flaviviridae, represents a major health problem in tropical and subtropical regions of the world. There are four serotypes, dengue virus types 1 to 4 (DENV-1 to DENV-4). DENV causes a wide range of symptoms, from mild febrile illness known as dengue fever (DF) to severe life-threatening illness, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Infection with one serotype induces life-long protection against homologous serotypes, but protection against other serotypes is short-lived. In secondary infection, cross-reactive, nonneutralizing antibodies bind to DENV. DENV-antibody complexes are taken up more efficiently by Fcγ receptor (FcγR)-expressing cells, and higher levels of viremia develop (5, 7, 10, 12, 15, 16). This phenomenon, known as antibody-dependent enhancement (ADE), is considered to be a risk factor for DHF and DSS.Protective immunity against DENV is best reflected by the presence of neutralizing antibody. High neutralizing antibody levels induced by primary infection are considered central in offering life-long protective immunity against the homologous serotypes. Thus, a vaccine against DENV infection is expected to induce high levels of neutralizing antibodies against all four serotypes. The plaque reduction neutralizing test (PRNT) is a widely accepted approach to measure the neutralizing activities of antibodies (14). PRNTs, which employ Vero, LLC-MK2, or BHK-21 cells (11, 14) are, however, limited to measuring neutralizing activities of viral infectivity in the absence of FcγR (1). It is possible that neutralizing antibody titers of anti-DENV antibodies induced by natural infection or by vaccines may differ when assayed in the presence of enhancing activity. The neutralizing antibody titers determined using FcγR-expressing BHK-21 cells may better reflect protective immunity, because the principal target cells of DENV are FcγR-expressing cells, such as monocytes (6). In the present study, we sought to determine if neutralizing antibody titers were at the same or different levels when BHK-21 cells and cell lines expressing FcγR were used as the assay cells.     

Poor Immune Responses of Newborn Rhesus Macaques to Measles Virus DNA Vaccines Expressing the Hemagglutinin and Fusion Glycoproteins

A vaccine that would protect young infants against measles could facilitate elimination efforts and decrease morbidity and mortality in developing countries. However, immaturity of the immune system is an important obstacle to the development of such a vaccine. In this study, DNA vaccines expressing the measles virus (MeV) hemagglutinin (H) protein or H and fusion (F) proteins, previously shown to protect juvenile macaques, were used to immunize groups of 4 newborn rhesus macaques. Monkeys were inoculated intradermally with 200 μg of each DNA at birth and at 10 months of age. As controls, 2 newborn macaques were similarly vaccinated with DNA encoding the influenza virus H5, and 4 received one dose of the current live attenuated MeV vaccine (LAV) intramuscularly. All monkeys were monitored for development of MeV-specific neutralizing and binding IgG antibody and cytotoxic T lymphocyte (CTL) responses. These responses were poor compared to the responses induced by LAV. At 18 months of age, all monkeys were challenged intratracheally with a wild-type strain of MeV. Monkeys that received the DNA vaccine encoding H and F, but not H alone, were primed for an MeV-specific CD8⁺ CTL response but not for production of antibody. LAV-vaccinated monkeys were protected from rash and viremia, while DNA-vaccinated monkeys developed rashes, similar to control monkeys, but had 10-fold lower levels of viremia. We conclude that vaccination of infant macaques with DNA encoding MeV H and F provided only partial protection from MeV infection.     

An Inactivated,Adjuvanted Whole Virion Clade 2.2 H5N1 (A/Chicken/Astana/6/05) Influenza Vaccine Is Safe and Immunogenic in a Single Dose in Humans

In this study, we assessed in humans the immunogenicity and safety of one dose (7.5 or 15 μg of hemagglutinin [HA]) of a whole-virion inactivated prepandemic influenza vaccine adjuvanted with aluminum hydroxide. The vaccine strain was made by reverse genetics from the highly pathogenic avian A/Chicken/Astana/6/05 (H5N1) clade 2.2 strain isolated from a dead bird in Kazakhstan. The humoral immune response was evaluated after a single vaccination by hemagglutination inhibition (HI) and microneutralization (MN) assays. The vaccine was safe and immunogenic, inducing seroconversion in 55% of the evaluated patients, with a geometric mean titer (GMT) of 17.1 and a geometric mean increase (GMI) of 3.42 after a dose of 7.5 μg in the HI test against the vaccine strain. The rate of seroconversion increased up to 70% when the dose of 15 μg was used. The percentages of individuals achieving anti-HA titers of ≥1:40 were 52.5% and 57.5% for the 7.5- and 15-μg dose groups, respectively. Similar results were obtained when antibodies were analyzed in an MN test. Substantial cross-neutralization titers (seroconversion in 35% and 52.5% of subjects in the two dose groups, respectively) were detected against heterologous clade 1 strain NIBRG14 (H5N1). Thus, one dose of this whole-virion prepandemic vaccine adjuvanted with aluminum has the potential to be effective against H5N1 viruses of different clades.     

Effect on cellular and humoral immune responses of the AS03 adjuvant system in an A/H1N1/2009 influenza virus vaccine administered to adults during two randomized controlled trials

The influence of AS03_A, a tocopherol oil-in-water emulsion-based adjuvant system, on humoral and cell-mediated responses to A/California/7/2009 H1N1 pandemic vaccine was investigated. In two observer-blind studies, a total of 261 healthy adults aged 18 to 60 years were randomized to receive either AS03_A-adjuvanted H1N1 vaccine containing 3.75 μg hemagglutinin (HA) or nonadjuvanted H1N1 vaccine containing 15 or 3.75 μg HA on days 0 and 21. Hemagglutination inhibition (HI) antibody and T-cell responses were analyzed up to day 42. A first dose of AS03_A-adjuvanted vaccine (3.75 μg HA) or nonadjuvanted vaccine (15 μg HA) induced HI responses of similar magnitudes that exceeded licensure criteria (e.g., 94 to 100% with titers of ≥40). A lower response following 3.75 μg HA without adjuvant was observed (73% with titers of ≥40). Following a second dose, geometric mean HI titers at day 42 were higher for AS03_A-adjuvanted vaccine (636 and 637) relative to nonadjuvanted vaccine (341 for 15 μg HA and 150 for 3.75 μg HA). Over the 42-day period, the increase in frequency of A/H1N1/2009-specific CD4⁺ T cells was significantly higher in the adjuvanted group than in the nonadjuvanted group. There was no evidence of correlation between baseline CD4⁺ T-cell frequencies and day 21 HI antibody titers, while there was some correlation (R = 0.35) between day 21 CD4⁺ T-cell frequencies and day 42 HI titers. AS03_A adjuvant enhanced the humoral and CD4⁺ T-cell-mediated responses to A/H1N1/2009 vaccine. Baseline A/H1N1/2009-specific CD4⁺ T-cell frequencies did not predict post-dose 1 antibody responses, but there was some correlation between post-dose 1 CD4⁺ T-cell frequencies and post-dose 2 antibody responses.     

A consensus envelope protein domain III can induce neutralizing antibody responses against serotype 2 of dengue virus in non-human primates

We have previously demonstrated that vaccination with a subunit dengue vaccine containing a consensus envelope domain III with aluminum phosphate elicits neutralizing antibodies against all four serotypes of dengue virus in mice. In this study, we evaluated the immunogenicity of the subunit dengue vaccine in non-human primates. After vaccination, monkeys that received the subunit vaccine with aluminum phosphate developed a significantly strong and long-lasting antibody response. A specific T cell response with cytokine production was also induced, and this correlated with the antibody response. Additionally, neutralizing antibodies against serotype 2 were detected in two of three monkeys. The increase in serotype-2-specific antibody titers and avidity observed in these two monkeys suggested that a serotype-2-biased antibody response occurs. These data provide evidence that a protective neutralizing antibody response was successfully elicited in non-human primates by the dengue subunit vaccine with aluminum phosphate adjuvant.     

Baboon model for West Nile virus infection and vaccine evaluation

Animal models that closely mimic the human condition are of paramount significance to study pathogenic mechanisms, vaccine and therapy scenarios. This is particularly true for investigations that involve emerging infectious diseases. Nonhuman primate species represent an alternative to the more intensively investigated rodent animal models and in a number of instances have been shown to represent a more reliable predictor of the human response to infection. West Nile virus (WNV) has emerged as a new pathogen in the Americas. It has a 5% fatality rate, predominantly in the elderly and immune compromised. Typically, infections are cleared by neutralizing antibodies, which suggests that a vaccine would be efficacious. Previously, only macaques had been evaluated as a primate model for WNV vaccine design. The macaques did not develop WNV disease nor express the full complement of IgG subclasses that is found in humans. We therefore explored baboons, which exhibit the similar four IgG subclasses observed in humans as a new model for WNV infection and vaccine evaluation. In this present report, we describe the experimental infection of baboons with WNV and test the efficacy of an inactivated WNV vaccination strategy. All experimentally infected animals developed transient viremia and subsequent neutralizing antibodies. Anti-WNV IgM antibodies peaked at 20 days post-infection. Anti-WNV IgG antibodies appeared later and persisted past 60 days. Prior vaccination with chemically inactivated virus induced neutralizing titers and a fast, high titer IgG recall response, which resulted in lower viremia upon challenge. This report is the first to describe the development of the baboon model for WNV experimental infection and the utility of this model to characterize the immunologic response against WNV and a candidate WNV vaccine.     

Immunogenicity of Mycoplasma pneumoniae

The immunogenicity of Mycoplasma pneumoniae for New Zealand white female rabbits was studied by using an immunization scheme which employed initial intramuscular immunization with vaccine in incomplete Freund adjuvant followed 3 weeks later by a series of five intravenous injections of fluid vaccine. Small doses of immunogen (15 to 150 μg of mycoplasmic protein per rabbit) gave rise to sera which contained antilipid complement-fixing antibody, produced one to three precipitin lines, but gave poor growth-inhibition on agar. Larger doses of immunogen (1.5 to 15 mg per rabbit) gave rise to sera which gave higher antilipid complement-fixing titers, four to eight precipitin lines, and good growth-inhibition. Doses smaller than 15 μg per rabbit failed to give rise to detectable antibody. Growth-inhibiting antibody was produced later than the other antibodies. The number of precipitin lines was controlled more critically by the quality of the antisera, as determined by the amount of immunogen, than by the quantity of serological test antigen. All sera which gave any precipitin lines produced a common precipitin line which stained for lipid.     

Functional Antibody Activity Elicited by Fractional Doses of Haemophilus influenzae Type b Conjugate Vaccine (Polyribosylribitol Phosphate–Tetanus Toxoid Conjugate)

We evaluated the functional activities of antibodies, serum bactericidal activity (SBA), and immunoglobulin G (IgG) antibody avidity indices, using sodium thiocyanate (NaSCN) elution, elicited after vaccination with fractional doses of the Haemophilus influenzae type b conjugate (polyribosylribitol phosphate [PRP] conjugated to tetanus toxoid [PRP-T]) vaccine. A cohort of 600 infants from the Dominican Republic were randomized to receive one of three regimens of the PRP-T vaccine at ages 2, 4, and 6 months: full doses (10 μg of PRP antigen), one-half doses (5.0 μg), and one-third doses (3.3 μg) (J. Fernandez et al., Am. J. Trop. Med. Hyg. 62:485–490, 2000). Sixty serum samples, collected at age 7 months, with ≥2.0 μg of anti-PRP IgG per ml were randomly selected for avidity determinations. Geometric mean IgG concentrations were 13, 14, and 17 μg/ml for infants who received the full-dose (n = 19), one-half-dose (n = 19), and one-third-dose (n = 22) regimens, respectively. SBA geometric mean titers (1/dilution) were 85.0, 82.0, and 76.1 in sera from infants receiving the full-, one-half-, and one-third-dose regimens, respectively. Avidity indices (mean ± standard error weighted average of NaSCN molar concentration × serum dilution factor) were 71.9 ± 9.4, 123.6 ± 26.8, and 150.9 ± 24.9 for the full-, one-half-, and one-third-dose regimens, respectively. Upon comparison, the only significant difference (P = 0.024) found was a greater avidity index for sera from infants receiving the one-third-dose regimen than for sera from infants receiving the the full-dose regimen. We conclude that fractional doses elicit similar functional antibody activities in infants with ≥2 μg of anti-PRP IgG per ml, corresponding to 89, 90, and 97% of infants receiving three doses of either the full concentration or one-half or one-third of the labeled concentration, respectively. This approach offers an alternative strategy for the prevention of H. influenzae type b disease in countries with limited resources.     

HIV-1 recombinant gp160 vaccine given in accelerated dose schedules

The purpose of this randomized, double-blind study was to test the safety and immunogenicity of an HIV-1_LAI recombinant gp160 (rgp160) vaccine in healthy, uninfected volunteers using accelerated dosing schedules. Thirty volunteers were randomly assigned to receive 50-μg doses of rgp160 in one of two immunization schedules. Group 1 received rgp160 at times 0, 1, 2 and 5 months; and group 2 received rgp160 at times 0, 1, 2, 3 and 4 months. The vaccine was safe and stimulated high levels of HIV-1 envelope-specific binding antibody and T cell memory. There was a trend (P < 0.10) suggesting neutralizing antibodies were better induced by the regimen incorporating a rest period before the final immunization in group 1 volunteers. Both accelerated immunization schedules induced immune responses at levels similar to or better than those achieved by four rgp160 vaccine injections given over 12-18 months in other studies.     

Evaluation of a recombinant Hc of Clostridium botulinum neurotoxin serotype F as an effective subunit vaccine

A new gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype F (FHc) was designed and completely synthesized with oligonucleotides. A soluble recombinant Hc of C. botulinum neurotoxin serotype F was highly expressed in Escherichia coli with this synthetic FHc gene. Subsequently, the purified FHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype F (BoNT/F). After the administration of FHc protein mixed with Freund adjuvant via the subcutaneous route, a strong protective immune response was elicited in the vaccinated mice. Mice that were given two or three vaccinations with a dosage of 1 or 10 μg of FHc were completely protected against an intraperitoneal administration of 20,000 50% lethal doses (LD₅₀) of BoNT/F. The BoNT/F neutralization assay showed that the sera from these vaccinated mice contained high titers of protective antibodies. Furthermore, mice were vaccinated once, twice, or three times at four different dosages of FHc using Alhydrogel (Sigma) adjuvant via the intramuscular route and subsequently challenged with 20,000 LD₅₀ of neurotoxin serotype F. A dose response was observed in both the antibody titer and the protective efficacy with increasing dosage of FHc and number of vaccinations. Mice that received one injection of 5 μg or two injections of ≥0.04 μg of FHc were completely protected. These findings suggest that the recombinant FHc expressed in E. coli is efficacious in protecting mice against challenge with BoNT/F and that the recombinant FHc subunit vaccine may be useful in humans.     

A plasmid encoding parts of the dengue virus E and NS1 proteins induces an immune response in a mouse model

A DENV-2 plasmid named pEII*EIII/NS1*, containing sequences encoding portions of the envelope protein that are potentially involved in the induction of neutralizing antibodies and a portion of the NS1 sequence that is involved in protection, is reported in this work. The synthesized subunit protein was recognized by human sera from infected patients and had the predicted size. The immunogenicity of this construct was evaluated using a mouse model in a prime-boost vaccination approach. The priming was performed using the plasmid pEII*EIII/NS1*, followed by a boost with recombinant full-length GST–E and GST–NS1 fusion proteins. The mice showed specific antibody responses to the E and NS1 proteins, as detected by ELISA, compared to the response of animals vaccinated with the parental plasmid. Interestingly, some animals had neutralizing antibodies. These results show that EII*, EIII and NS1* sequences could be considered for the design of a recombinant subunit vaccine against dengue disease.     

Protection against dengue virus by non-replicating and live attenuated vaccines used together in a prime boost vaccination strategy

A new vaccination strategy for dengue virus (DENV) was evaluated in rhesus macaques by priming with tetravalent purified inactivated virus (TPIV) or tetravalent plasmid DNA vaccines expressing the structural prME gene region (TDNA) then boosting 2 months later with a tetravalent live attenuated virus (TLAV) vaccine. Both vaccine combinations elicited virus neutralizing (N) antibodies. The TPIV/TLAV combination afforded complete protection against DENV 3 challenge at month 8. In a second experiment, priming with TPIV elicited N antibodies against all four serotypes (GMT 1:28 to 1:43). Boosting with TLAV led to an increase in the GMT for each serotype (1:500 to 1:1200 for DENVs 1, 3, and 4, and greater than 1:6000 for DENV 2), which declined by month 8 (GMT 1:62 for DENV 3, 1:154 for DENV 1, 1:174 for DENV 4, and 1:767 for DENV 2). After challenge with each one of the four DENV serotypes, vaccinated animals exhibited no viremia but showed anamnestic antibody responses to the challenge viruses.     

Dengue-2 Vaccine: Viremia and Immune Responses in Rhesus Monkeys

Studies were undertaken in Indian rhesus monkeys (Macaca mulatta) to determine the safety, potency, immunogenicity, and mosquito infectivity of a small-plaque, temperature-sensitive variant of dengue type 2 (DEN-2) virus, a vaccine candidate. Fifteen monkeys were inoculated subcutaneously with the vaccine virus, ten receiving 10^3.1 plaque-forming units (PFU) and five receiving 10^4.5 PFU. After primary immunization, viremia was detected in only one monkey, a recipient of the higher dose of vaccine. The recovered virus had the same growth characteristics as the vaccine strain. Aedes aegypti mosquitoes did not become infected when they were allowed to feed on monkeys that received the lower dose of vaccine. As expected, the immunization produced no evidence of illness in any of the animals. A dose response to vaccine was detected; all five of the high-dose recipients developed neutralizing antibodies, whereas only five of ten low-dose recipients did so. In both groups, neutralizing antibody was often transient. Its presence at 30 days did not always correlate with protection from viremia in those animals challenged 4 to 6 months after vaccination with wild-type DEN-2 virus. However, immunized animals developed anamnestic antibody responses after challenge, and none demonstrated adverse effects to infection. Reimmunization of monkeys 4 months after primary immunization led to the production of low-titered but persistent neutralizing antibody which protected the animals from a wild-type virus challenge.     

Development of a Recombinant Xenogeneic Tumor Necrosis Factor Alpha Protein Vaccine To Protect Mice from Experimental Colitis

Previous studies have highlighted the efficacy of tumor necrosis factor alpha (TNF-α) inhibitors, including monoclonal antibodies and soluble receptors, in the treatment and management of intestinal bowel disease (IBD). However, because of the immunogenicity of xenogeneic TNF-α inhibitors, antidrug antibodies (ADAs) can be triggered after repeated administration. An alternative way to target TNF-α is active immunization to elicit the production of high titers of neutralizing antibodies. In this study, we prepared a xenogeneic TNF-α protein vaccine and studied the protective effects in experimental colitis models. The xenogeneic TNF-α protein vaccine could overcome self-tolerance and induce TNF-α-specific neutralizing antibody. Moreover, the xenogeneic TNF-α protein vaccine could protect mice from acute and chronic colitis induced by dextran sodium sulfate (DSS). One possible explanation for this protective effect is the production of TNF-α-specific neutralizing antibody, which absorbed the biological activity of mouse TNF-α (mTNF-α) and failed to induce T lymphocyte apoptosis. In summary, use of the xenogeneic TNF-α protein vaccine may be a potent therapeutic strategy for IBD.     

Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain from Pichia pastoris as a Recombinant Vaccine Candidate

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(H_c)], expressed in Pichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(H_c) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 μg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD₅₀) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 μg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD₅₀ of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of ≥0.5 μg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of ≥1,600, as measured by ELISA, survived, but only 6 of 42 mice with titers of ≤100 survived. This work shows that the purified BoNT/A(H_c) produced was a highly effective immunogen, able to protect against a high challenge dose of neurotoxin.     

Simultaneous Quantitation of Antibodies to Neutralizing Epitopes on Virus-Like Particles for Human Papillomavirus Types 6, 11, 16, and 18 by a Multiplexed Luminex Assay

Several different methods have been developed to quantitate neutralizing antibody responses to human papillomaviruses (HPVs), including in vivo neutralization assays, in vitro pseudoneutralization assays, competitive radioimmunoassays (cRIAs), and enzyme-linked immunosorbent assays. However, each of these techniques possesses one or more limitations that preclude testing large numbers of patient sera for use in natural history studies and large vaccine clinical trials. We describe here a new multiplexed assay, by using the Luminex Laboratory MultiAnalyte Profiling (LabMAP3) assay system, that can simultaneously quantitate neutralizing antibodies to human papillomavirus types 6, 11, 16, and 18 in 50 μl of serum. The HPV-Luminex competitive immunoassay measures titers of polyclonal antibodies in serum capable of displacing phycoerythrin-labeled detection monoclonal antibodies binding to conformationally sensitive, neutralizing epitopes on the respective virus-like particles. This competitive Luminex immunoassay was found to be as sensitive, accurate, and precise as the currently used cRIAs. An effective HPV vaccine will most likely require several distinct genotypes to protect against multiple cancer causing papillomaviruses. The HPV-Luminex immunoassay should prove to be a useful tool in simultaneously quantitating antibody immune responses to multiple HPV genotypes for natural history infection studies and for monitoring the efficacy of prospective vaccines.     

Immunogenicity of whole cell and outer envelope leptospiral vaccines in hamsters

The immunogenicity of leptospiral whole cell (WC) and outer envelope (OE) vaccines from virulent and avirulent strains was compared in hamsters. The 50% protective dose against death (PD_50D) and kidney infection (PD_50K) was evaluated for serotypes icterohaemorrhagiae, canicola, pomona, and grippotyphosa. The OE and WC of virulent strains possessed greater immunogenicity than the OE and WC of avirulent strains. More vaccine (<0.1 to 11.0 μg [dry weight] per hamster) was required for the PD_50K than for the PD_50D (<0.1 to 1.7 μg [dry weight] per hamster). The OE from virulent strains of these four serotypes plus hardjo were combined to form a pentavalent OE vaccine which proved to be satisfactory in immunogenic potency. Duration of immunity was not evaluated.     

Validation of autoradiography for recognition of antigen-binding lymphocytes in blood and lymphoid tissues: quantitation and specificity of binding

Antigen-binding lymphocytes were recognized by their reaction with radioiodine labelled antigens such as flagellin and haemocyanin. Counts varied according to the antigen and species studied. For flagellin, counts in human blood of antigen-binding lymphocytes (mean ± 1 SD per 1000 lymphocytes) were 19·0±3·0, and in foetal thymus 18·2±5·0 and spleen 3·5±0·5. Results depended on contact time of cells with antigen, concentration of antigen, autoradiographic exposure, presence of natural antibody and antibody levels after immunization. Antigen-binding lymphocytes in blood were not antibody-producing cells. The specificity of the antigen-binding reaction was shown by exposing lymphocytes to 0·5 μg of two antigenically distinct flagellins; there was a 67–100% increase in the counts in contrast to the 20–45% increase on doubling the dose (0·5 μg to 1 μg) of flagellin from Salmonella adelaide. Cytophilic antibody as the cause of antigen binding was excluded.

The binding of flagellin to lymphocytes was prevented by anti-human IgM and light chain antisera, but not anti-human IgG sera. The binding of labelled flagellin was prevented by unlabelled flagellin but 100 times more was needed for blood lymphocytes than thymocytes. It is inferred that thymocytes, T cells, have considerably fewer receptors than most β lymphocytes detectable in blood.

Using standardized conditions, radiolabelled antigen binding provides a reproducible, immunologically specific and flexible technique allowing study of the nature and role of antigen-binding cells and cell surface receptors.

     

Mechanism of induction of immunological tolerance: VI. Tolerance induction following thoracic duct drainage or treatment with anti-lymphocyte serum*

Adult Wistar rats were injected with flagellin from Salmonella adelaide three times weekly for different periods of time. With the range of doses 100 fg to 100 μg (fg, femtograms), varying levels of immune responsiveness were demonstrable following challenge, but not one of the doses induced complete immunological tolerance, although a reduced antibody response was elicited in rats which received injections of 100 μg of flagellin.

In contrast to this finding, complete tolerance could be demonstrated following challenge if rats, previously drained of lymph and lymphocytes from the thoracic duct for 5 days, were injected with either 100 μg of flagellin three times weekly or with 1 μg/g body weight/day for 6 weeks. Similarly, anti-lymphocyte serum treatment prior to the injection of antigen resulted in partial tolerance in adult rats and nearly complete tolerance in adult C57BL/Brad mice. The primary response to flagellin of C57BL mice was abrogated if ALS was administered prior to but not after the injection of antigen. The implications of these findings are discussed.