Comparison of Intranasal and Intramuscular Immunization against Human Immunodeficiency Virus Type 1 with a DNA-Monophosphoryl Lipid A Adjuvant Vaccine

We compared immune responses to intranasal and intramuscular DNA vaccinations against human immunodeficiency virus type 1 with monophosphoryl lipid A (MPL) used as an adjuvant. Both routes of vaccination resulted in similar levels of cell-mediated immunity, but the intestinal secretory immunoglobulin A response was higher following intranasal immunization than after intramuscular immunization. MPL demonstrated its adjuvanticity in vaccination by both routes.     

Both Immunization with Protein and Recombinant Vaccinia Virus Can Stimulate CTL Specific for the E7 Protein of Human Papilloma Virus 16 in H-2d Mice

The transforming protein E7 of human papilloma virus type 16 can stimulate cytotoxic T lymphocytes (CTL) which can protect experimental animals against growth of E7 expressing tumour cells. In this study we compared CTL responses in mice immunized with either E7 protein in MF59 adjuvant or with recombinant vaccinia virus expressing E7 (Vac-E7). We have chosen H-2^d mice because no E7-specific CTL responses have been described in this MHC haplotype. Immunization of these mice with Vac-E7 generated CTL which lysed target cells infected with Vac-E7 or transfected with the E7 gene. CTL from mice immunized with E7 protein in MF59 adjuvant showed specificity for the same target cells. Antibody blocking experiments revealed that both immunization with Vac-E7 and E7 protein stimulated CD8⁺ effector CTL. The find specificity of CTL induced by the two immunization protocols was similar. A major CTL epitope was mapped to the carboxyl terminal amino acids 48–98 of the E7 protein. Peptide isolation from E7 expressing cells followed by HPLC separation indicated that CTL induced by immunization with protein and Vac-E7 recognized the same HPLC purified peptide fractions. Together, the study suggests that vaccines based on protein can activate CTL with similar fine specificity to CTL induced by vaccines based on recombinant vaccinia virus.     

A Three-Dose Intramuscular Injection Schedule of Anthrax Vaccine Adsorbed Generates Sustained Humoral and Cellular Immune Responses to Protective Antigen and Provides Long-Term Protection against Inhalation Anthrax in Rhesus Macaques

A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r² = 0.89 for log₁₀-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-γ) and interleukin-4 (IL-4) CD4⁺ cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.     

Protection of Sheep against Rift Valley Fever Virus and Sheep Poxvirus with a Recombinant Capripoxvirus Vaccine

Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.Rift Valley fever (RFV) virus (RVFV) is a mosquito-borne member of the genus Phlebovirus, family Bunyaviridae. It is widely distributed in Africa, causing endemic and epidemic disease in both humans and livestock, including sheep, cattle, and goats. RVF was first described in Kenya and was shown to be caused by a filterable virus transmissible via blood (9). Acute RVF in lambs is characterized by fever and death within 24 to 48 h of being detected (43). Signs in adult sheep include fever, mucopurulent nasal discharge, hemorrhagic diarrhea, and abortion in pregnant ewes (43). RVFV can be transmitted from infected sheep to humans, particularly when humans are exposed to aborted sheep fetuses and blood.Attenuated live RVFV vaccines are available for use in livestock. A mutagen-attenuated RVFV vaccine induces protective immune responses in lambs and appears to be safe (25); however, other studies documented teratogenic effects on lambs from vaccinated pregnant ewes similar to those caused by the attenuated RVFV strain Smithburn (18). An inactivated RVFV vaccine induces neutralizing antibody responses in humans (33), and its use in sheep would not induce teratogenic effects or abortions. However, the inactivated vaccine requires 3 doses (33) and is expensive to produce. Efforts to make RVFV vaccines without these disadvantages include an attenuated RVFV developed by reverse genetics and lacking the NSs and NSm genes (4) and other new-generation RVFV vaccines (reviewed in reference 19) that protect mice against virus challenge (7, 16, 24, 27).The middle (M) RNA segment of the RVFV genome encodes the viral glycoproteins Gn and Gc (8, 20), and recombinant vaccinia virus expressing these glycoproteins induces neutralizing antibody and protective immunity to RVFV in mice (7). Vaccinia virus is safe for animals, but there is some risk to humans, as it was reported previously to spread from human vaccinees to contacts (28, 55) and to cause serious clinical disease in human immunodeficiency virus-infected patients (36). Although modified vaccinia virus Ankara is a safer alternative for humans (6, 57), there are animal poxviruses with naturally restricted host ranges for vaccine vectors in animals (1, 13, 30, 31, 40, 46, 47, 52, 53).For ruminants, the genus Capripoxvirus (CPV) of the family Poxviridae has been an effective recombinant vector to induce protective immunity against several other viruses (3, 17, 29, 32, 40, 41, 51). This genus has three closely related species causing sheep pox, goat pox, and lumpy skin disease (LSD) of cattle. A recombinant LSD vaccine expressing the Gn and Gc glycoproteins of RVFV induced protection against RVFV challenge in mice (52, 53) and sheep (52). The three species of CPV have 96 to 97% nucleotide identity (49) and are restricted to ruminants, with no evidence of human infections (10, 11). Furthermore, attenuated CPV vaccines are in use in Africa and the Middle East to control ruminant poxvirus disease (11, 21). The use of a CPV vector to deliver virus vaccines to ruminants also induces immunity to the CPV vector, thus increasing the valence of the vaccine (3, 17, 39, 40). We report here the construction of a recombinant CPV that expresses the RVFV Gn and Gc glycoproteins and induces protective immunity against RVFV and sheep poxvirus (SPV) challenge in sheep.     

Partial Protection against Plasmodium vivax Blood-Stage Infection in Saimiri Monkeys by Immunization with a Recombinant C-Terminal Fragment of Merozoite Surface Protein 1 in Block Copolymer Adjuvant

Merozoite surface protein 1 is a candidate for blood-stage vaccines against malaria parasites. We report here an immunization study of Saimiri monkeys with a yeast-expressed recombinant protein containing the C terminus of Plasmodium vivax merozoite surface protein 1 and two T-helper epitopes of tetanus toxin (yP₂P₃₀Pv200₁₉), formulated in aluminum hydroxide (alum) and block copolymer P1005. Monkeys immunized three times with yP₂P₃₀Pv200₁₉ in block copolymer P1005 had significantly higher prechallenge titers of immunoglobulin G (IgG) antibodies against the immunogen and asexual blood-stage parasites than those immunized with yP₂P₃₀Pv200₁₉ in alum, antigen alone, or phosphate-buffered saline (PBS) (P < 0.05). Their peripheral blood mononuclear cell proliferative responses to immunogen stimulation 4 weeks after the second immunization were also significantly higher than those from the PBS control group (P < 0.05). Upon challenge with 100,000 asexual blood-stage parasites 5 weeks after the last immunization, monkeys immunized with yP₂P₃₀Pv200₁₉ in block copolymer P1005 had prepatent periods longer than those for the control alone group (P > 0.05). Three of the five animals in this group also had low parasitemia (peak parasitemia, ≤20 parasites/μl of blood). Partially protected monkeys had significantly higher levels of prechallenge antibodies against the immunogen than those unprotected (P < 0.05). There was also a positive correlation between the prepatent period and titers of IgG antibodies against the immunogen and asexual blood-stage parasites and a negative correlation between accumulated parasitemia and titers of IgG antibodies against the immunogen (P < 0.05). These results indicate that when combined with block copolymer and potent T-helper epitopes, the yeast-expressed P₂P₃₀Pv200₁₉ recombinant protein may offer some protection against malaria.     

Prime-Boost Immunization of Codon Optimized HIV-1 CRF01_AE Gag in BCG with Recombinant Vaccinia Virus Elicits MHC Class I and II Immune Responses in Mice

The HIV-1 CRF01_AE gag gene was modified by codon restriction for Mycobacterium spp. and transformed into BCG; and it was designated as rBCG/codon optimized gagE. This produced 11 fold higher HIV-1 gag protein expression than the recombinant native gene rBCG/HIV-1gagE. In mice, CTL activity could be induced either by a single immunization of the codon optimized construct or by using it as a priming antigen in the prime-boost modality with recombinant Vaccinia virus expressing native HIV-1 gag. Specific secreted cytokine responses were also investigated. Only when rBCG gag was codon optimized did the prime-boost immunization produce significantly enhanced IFN-γ and IL-2 secretion indicating recognition via CD4+ and CD8+ T cells, and these responses seemed to be codon optimized immunogen dose-responsive. On contrary, the prime-boost vaccination using an equal amount of native rBCG/HIV-1gagE instead, or a single rBCG/codon optimized gagE immunization, had no similar effect on the cytokine secretion. These findings suggest that the use of recombinant codon BCG construct with recombinant Vaccinia virus encoding CRF01_AE gag as the prime-boost HIV vaccine candidate, will induce CD4+ Th1 and CD8+ T cell cytokine secretions in addition to enhancing CD8+ CTL response.     

Protection against Cholera Toxin after Oral Immunization is Thymus-Dependent and Associated with Intestinal Production of Neutralizing IgA Antitoxin

We studied whether gut mucosal IgA antitoxin production as well as the acquired protection against cholera toxin (CT) after oral immunization with CT are both thymus-dependent immune manifestations. In contrast to normal BALB/c mice, nude, athymic mice did not respond to oral immunizations with CT with either IgA antitoxin-producing cells (SFC) in the lamina propria or protection against challenge with CT in ligated intestinal loops. However, when nude mice were first reconstituted by grafting of syngeneic thymus glands, both IgA antitoxin SFC in the lamina propria and protection were stimulated by oral immunizations with CT and the response were of similar magnitude to those of normal mice after immunizations. During in vitro culture, isolated lamina propria lymphocytes from immunized but not from control mice concomitantly and proportionally produced IgA antitoxin and CT-neutralizing activity. We conclude that intestinal antitoxin formation and protection against toxin challenge after oral immunization with CT are both critically thymus-dependent and therefore likely to be under T-cell control.     

Immunization with a Combination of Integral Chlamydial Antigens and a Defined Secreted Protein Induces Robust Immunity against Genital Chlamydial Challenge

We have previously demonstrated the efficacy of recombinant chlamydial protease-like activity factor (rCPAF; a secreted chlamydial protein) in inducing antigen-specific CD4⁺ T cell/gamma interferon (IFN-γ)-mediated but not antibody-mediated chlamydial clearance and reduction of upper genital tract (UGT) pathological sequelae. Since chlamydial integral antigens may induce neutralizing antibody protection, we further evaluated induction of protective immunity using a combination of rCPAF and UV-inactivated chlamydial elementary bodies (UV-EB) against vaginal chlamydial challenge in comparison to immunization with the individual components or live EB. The rCPAF-UV-EB immunization induced a significantly enhanced anti-UV-EB cellular and antibody response and a reduced anti-CPAF cellular and antibody response, compared to immunization with the respective individual components. Moreover, vaccination with UV-EB and rCPAF-UV-EB induced serum antibodies that neutralized chlamydial infectivity. The rCPAF-UV-EB immunization resulted in a significant reduction of vaginal chlamydial shedding and induced earlier bacterial clearance than vaccination of mice with the individual components. Importantly, the UGT sequelae were significantly reduced in mice immunized with rCPAF or rCPAF-UV-EB, but not in those immunized with UV-EB alone, and approached the levels of protection induced by live EB. These results collectively suggest that a combination of neutralizing antibodies induced by integral chlamydial antigens and cell-mediated responses induced by secreted proteins such as CPAF induces optimal protective immunity against genital chlamydial infections.There is currently no licensed vaccine against Chlamydia trachomatis, the leading cause of bacterial sexually transmitted disease worldwide (2, 16). We have previously demonstrated the efficacy of recombinant chlamydial protease-like activity factor (rCPAF) in inducing protective immunity against genital chlamydial challenge (23). Immunization using rCPAF with a T helper 1 (Th1)-type adjuvant induces significantly enhanced bacterial clearance and robust protection against upper genital tract (UGT) pathology following vaginal challenge with homologous or heterologous serovars/species of Chlamydia (5, 6, 23). The high degree of cross-serovar/species protection against UGT sequelae highlights the importance of further characterizing the potential of rCPAF as a component of an antichlamydial vaccine for humans (25). rCPAF-vaccinated mice display significant protection against UGT chlamydial sequelae and clear the bacteria with significantly accelerated kinetics, achieving complete clearance by day 18 (day 30 in mock-vaccinated mice) after challenge. However, vaginal bacterial shedding in rCPAF-vaccinated mice is comparable to the level for mock-vaccinated controls during the initial week after challenge (6, 23). Such enhanced clearance kinetics, in the absence of resistance to infection, may be attributed to the dependence of the protective response on gamma interferon (IFN-γ)-producing CPAF-specific CD4⁺ T cells (15), a limited role for anti-CPAF antibody (22), and the restriction of CPAF to replicating reticulate bodies.Chlamydia muridarum infection in mice induces a high level of protective immune responses, including a certain degree of resistance to reinfection, mediated by robust IFN-γ-producing CD4⁺ T cell responses (4, 11-13, 16, 17, 20, 28-31, 34) and antibodies (16,18-20). A single immunogenic subunit that induces protective immunity comparable to that induced by live, replicating chlamydial organisms has yet to be identified (2, 16, 25). The immunogenic proteins that serve as targets for antibody and T cell responses may be broadly categorized, albeit with some overlap, as proteins that are integral to the chlamydial organism and those that are secreted from the organism, respectively. Specifically, proteins integral to the chlamydial organism would likely serve as targets for neutralizing infectivity extracellularly but may not be candidates of choice for eliciting T cell-mediated killing, due to the sturdy inclusion membrane barrier between the organisms and antigen-presentation pathways during the intracellular developmental cycle (25). On the other hand, secreted proteins such as CPAF are not present on the infectious chlamydial elementary body (EB) and therefore would not be expected to serve as targets for neutralizing chlamydial infectivity (25). However, proteins secreted into the host cytosol, and thereafter into extracellular compartments, may serve as exogenous antigens and a suitable target for CD4⁺ T cell-mediated effector responses (25, 37). Thus, it would appear that both integral and secreted proteins of Chlamydia may serve as targets for complementary immune responses and that the greatest potential for successful vaccination could be derived by combining them in a multisubunit vaccine.In this study, we compared the protective immunities induced by intranasal (i.n.) immunization with rCPAF, UV-inactivated EBs (UV-EB), rCPAF-UV-EB, or live EB against genital C. muridarum challenge in female BALB/c mice. The combination of integral and secreted proteins enhanced protective immunity compared to the individual components and approached the high level of protection induced by live, replicating chlamydial organisms.     

Long-Term Cholera Antitoxin Memory in the Gut can be Triggered to Antibody Formation Associated with Protection within Hours of an Oral Challenge Immunization

A local mucosal immunological memory that could be efficiently triggered to protective antibody formation on renewed antigen exposure might account for the several-year long protection against reinfection and disease seen in individuals after cholera disease. The duration and other functional aspects of gut mucosal immunological memory to the cholera toxin (CT), which is the key pathogenic factor in cholera, were examined in mice. Six months or even 2 years after an initial series of oral immunizations with CT a single repeat oral exposure to CT in submicrogram amounts evoked a brisk IgA antitoxin response in the lamina propria. A three-fold increase in IgA antitoxin-producing cells (SFC) was evident within 16 h, with a further rise in SFC numbers over the next several days. The anamnestic gut mucosal IgA antitoxin response was associated with a substantial increase in protection against challenge of intestinal loops with CT. The rapid increase in IgA antitoxin SFC in the gut is believed to reflect memory cells dispersed in the gut mucosa which can be rapidly triggered into antitoxin formation by antigen encounter in vivo and such cells could clearly be responsible for the long-term immunity seen after cholera disease or oral vaccination.     

Recombinant GRA4 or ROP2 Protein Combined with Alum or the gra4 Gene Provides Partial Protection in Chronic Murine Models of Toxoplasmosis

The efficacy of vaccination with Toxoplasma gondii recombinant GRA4 (rGRA4) and ROP2 (rRPO2) proteins and a mix of both combined with alum were evaluated in C57BL/6 and C3H mice. In C57BL/6 mice, rGRA4 and rGRA4-rROP2 immunizations generated similar levels of immunoglobulin G1 (IgG1) and IgG2a isotypes against GRA4, whereas immunizations with rROP2 and the mix induced a predominant IgG1 production against ROP2. All groups of C3H vaccinated mice exhibited higher levels of IgG1 than IgG2a. rGRA4-stimulated splenocytes from vaccinated mice produced primarily gamma interferon while those stimulated with rROP2 produced interleukin-4. Challenge of rGRA4- or rGRA4-rROP2-vaccinated mice from both strains with ME49 cysts resulted in fewer brain cysts than the controls, whereas vaccination with rROP2 alone only conferred protection to C3H mice. Immunization with a plasmid carrying the entire open reading frame of GRA4 showed a protective level similar to that of rGRA4 combined with alum. These results suggest that GRA4 can be a good candidate for a multiantigen anti-T. gondii vaccine based on the use of alum as an adjuvant.     

Immunization of Saimiri sciureus Monkeys with a Recombinant Hybrid Protein Derived from the Plasmodium falciparum Antigen Glutamate-Rich Protein and Merozoite Surface Protein 3 Can Induce Partial Protection with Freund and Montanide ISA720 Adjuvants

The immunogenicity and efficacy of a hybrid recombinant protein derived from the N-terminal end of the glutamate-rich protein (GLURP) and the C-terminal portion of the merozoite surface protein 3 (MSP3) of Plasmodium falciparum was evaluated in Saimiri sciureus monkeys. The GLURP/MSP3 hybrid protein, expressed in Lactococcus lactis, was administered in association with alum, Montanide ISA720, or complete or incomplete Freund adjuvant (CFA/IFA) in groups of five animals each. The three formulations were shown to be immunogenic, but the one with alum was shown to be weak compared to the other two, particularly CFA/IFA, which provided very high antibody titers (enzyme-linked immunosorbent assay titers of >3,000,000 and immunofluorescence antibody test titers of 6,400). After a challenge infection with P. falciparum FUP strain, all five monkeys from the GLURP/MSP3-alum group showed a rapid increase in parasitemia, reaching 10% and were treated early. The two monkeys with the highest antibody titers in group GLURP/MSP3-Montanide ISA720 had a delay in the course of parasitemia and were treated late due to a low hematocrit. In the GLURP/MSP3-CFA/IFA group, parasitemia remained below this threshold in four of the five animals and, after it reached a peak, parasitemia started to decrease and monkeys were treated late. When all animals were grouped according to the outcome, a statistically significant association between high antibody titers and partial protection was observed. The challenge infection boosted the antibody titers, and the importance of this event for vaccine efficacy in areas where this parasite is endemic is discussed. In conclusion, these data suggest that GLURP and MSP3 can induce protection against malaria infection if antibodies are induced at properly high titers.     

Replicating Adenovirus-Simian Immunodeficiency Virus (SIV) Vectors Efficiently Prime SIV-Specific Systemic and Mucosal Immune Responses by Targeting Myeloid Dendritic Cells and Persisting in Rectal Macrophages,Regardless of Immunization Route

Although priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, followed by HIV/SIV envelope boosting, has proven highly immunogenic, resulting in protection from SIV/simian-human immunodeficiency virus (SHIV) challenges, Ad5hr recombinant distribution, replication, and persistence have not been examined comprehensively in nonhuman primates. We utilized Ad5hr-green fluorescent protein and Ad5hr-SIV recombinants to track biodistribution and immunogenicity following mucosal priming of rhesus macaques by the intranasal/intratracheal, sublingual, vaginal, or rectal route. Ad recombinants administered by all routes initially targeted macrophages in bronchoalveolar lavage (BAL) fluid and rectal tissue, later extending to myeloid dendritic cells in BAL fluid with persistent expression in rectal mucosa 25 weeks after the last Ad immunization. Comparable SIV-specific immunity, including cellular responses, serum binding antibody, and mucosal secretory IgA, was elicited among all groups. The ability of the vector to replicate in multiple mucosal sites irrespective of delivery route, together with the targeting of macrophages and professional antigen-presenting cells, which provide potent immunogenicity at localized sites of virus entry, warrants continued use of replicating Ad vectors.     

The Vaccinia Virus E3L Protein Interacts with SUMO-1 and Ribosomal Protein L23a in a Yeast Two Hybrid Assay

We report the results of a two-hybrid study which identified clones from a HeLa cDNA library that interact with the vaccinia virus protein E3L. These clones encode the nuclear protein SUMO-1 (also known as PIC-1, sentrin or GMP-1); the cytoplasmic ribosomal protein L23a; and a small peptide sequence of unknown significance.     

Prolonged Infection in Rhesus Macaques with Simian Immunodeficiency Virus (SIVmac239) Results in Animal-Specific and Rarely Tissue-Specific Selection ofnefVariants

We analyzed the sequence ofnefgenes from different tissues of three rhesus macaques that had been infected with molecularly cloned SIV_mac239 for 88 to 92 weeks. Comparison of the predicted amino acid sequences revealed that each macaque had selected out specific amino acid substitutions and that most of this variation (70%) was confined to four regions, amino acids 39 to 75, 90 to 105, 153 to 167, and 191 to 217, comprising 36% of the protein. Thenefgenes in these animals underwent extensive genetic variation with average nucleotide and amino acid substitution rates varying from 0.86 to 2.84% and 2.47 to 6.27%, respectively, although tissue-specific selection ofnefvariants occurred in only 1 of 14 tissues examined in this study. Comparison of the rate of nucleotide and amino acid substitutions in thenefgenes to those previously reported in theenvin the central nervous system (CNS) and lymph node (LN) revealed that the predicted amino acid substitution rates for Nef were much higher than for the gp120 region ofenvin the CNS and LN tissues for one macaque. In the two other macaques, the predicted amino acid substitution rates were similar between these two proteins in LN tissues, but the amino acid substitution rates in Nef were significantly higher than in the gp120 from the CNS. Comparison of the nucleotide substitutions in the region of overlap between theenvand thenefrevealed that approximately 83% of the nucleotide substitutions in this area resulted in a Nef amino acid sequence change, 26% of the nucleotide substitutions resulted in a gp41 amino acid change, and 9.5% of nucleotide substitutions resulted in amino acid sequence changes in both proteins, suggesting a preference for the selection of amino acid substitutions in the Nef in these animals. Our results indicate that in animals infected with SIV_mac239 for prolonged periods, variation in thenefoccurs at rates similar to or exceeding that observed for theenvgene.     

A Chimeric Influenza Virus Expressing an Epitope of Outer Membrane Protein F of Pseudomonas aeruginosa Affords Protection against Challenge with P. aeruginosa in a Murine Model of Chronic Pulmonary Infection

The ability of a chimeric influenza virus containing, within the antigenic B site of its hemagglutinin, an 11-amino-acid (AEGRAINRRVE) insert from the peptide 10 epitope of outer membrane (OM) protein F of Pseudomonas aeruginosa to serve as a protective vaccine against P. aeruginosa was tested by using the murine chronic pulmonary infection model. Mice immunized with the chimeric virus developed antibodies that reacted in an enzyme-linked immunosorbent assay with peptide 10, with purified protein F, and with whole cells of various immunotype strains of P. aeruginosa but failed to react with a protein F-deficient strain of P. aeruginosa. The chimeric-virus antisera reacted specifically with protein F alone when immunoblotted against proteins extracted from cell envelopes of each of the seven Fisher-Devlin immunotype strains and had significantly greater in vitro opsonic activity for P. aeruginosa than did antisera from wild-type influenza virus-immunized mice. Subsequent to intratracheal challenge with agar-encased cells of P. aeruginosa, chimeric-virus-immunized mice developed significantly fewer severe lung lesions than did control mice immunized with the wild-type influenza virus. Furthermore, the chimeric influenza virus-immunized group had a significantly smaller percentage of mice with >5 × 10³ CFU of P. aeruginosa in their lungs upon bacterial quantitation than did the control group. These data indicate that chimeric influenza viruses expressing epitopes of OM protein F warrant continued development as vaccines to prevent pulmonary infections caused by P. aeruginosa.     

Impaired Generation of Anti-Viral Cytotoxicity against Lymphocytic Choriomeningitis and Vaccinia Virus in Mice Treated with CD4-Specific Monoclonal Antibody

The role of CD4+ helper T cells in induction of anti-viral cytotoxic T-cell response was investigated by treating normal and thymectomized C57B1/6 mice with CD4-specific monoclonal antibodies (MoAb). In CD4-specific MoAb-treated mice infected with Vaccinia or lymphocytic choriomeningitis virus (LCMV), cytotoxic T-cell activity was 5-15 times lower than in normal controls when measured in a 51Cr release assay and computed as lytic units 6 and 8 days respectively after virus inoculation. This difference in the levels of effector T-cell activities did not reflect slower kinetics of cytotoxic T-cell induction in antibody-treated versus control mice, since it was also obvious at 8 days after infection for Vaccinia virus and 10 and 12 days after inoculation with LCMV. CD4-specific MoAb-induced inhibition of cytotoxic T-cell responses in vivo was seen up to 150 days after treatment in thymectomized mice. However, no significant suppressive effect of the same antibody treatment on T-cell cytotoxicity could be observed in animals treated on day 3 or later after infection with Vaccinia virus. Injection of CD4-depleted mice with recombinant interleukin 2 (rIL-2) partially corrected the impaired virus-specific cytotoxic T-cell response, suggesting that IL-2 supply may be limiting in mice lacking T helper cells.     

Intranasal Vaccination with Replication-Defective Adenovirus Type 5 Encoding Influenza Virus Hemagglutinin Elicits Protective Immunity to Homologous Challenge and Partial Protection to Heterologous Challenge in Pigs

Influenza A virus (IAV) is widely circulating in the swine population and causes significant economic losses. To combat IAV infection, the swine industry utilizes adjuvanted whole inactivated virus (WIV) vaccines, using a prime-boost strategy. These vaccines can provide sterilizing immunity toward homologous virus but often have limited efficacy against a heterologous infection. There is a need for vaccine platforms that induce mucosal and cell-mediated immunity that is cross-reactive to heterologous viruses and can be produced in a short time frame. Nonreplicating adenovirus 5 vector (Ad5) vaccines are one option, as they can be produced rapidly and given intranasally to induce local immunity. Thus, we compared the immunogenicity and efficacy of a single intranasal dose of an Ad5-vectored hemagglutinin (Ad5-HA) vaccine to those of a traditional intramuscular administration of WIV vaccine. Ad5-HA vaccination induced a mucosal IgA response toward homologous IAV and primed an antigen-specific gamma interferon (IFN-γ) response against both challenge viruses. The Ad5-HA vaccine provided protective immunity to homologous challenge and partial protection against heterologous challenge, unlike the WIV vaccine. Nasal shedding was significantly reduced and virus was cleared from the lung by day 5 postinfection following heterologous challenge of Ad5-HA-vaccinated pigs. However, the WIV-vaccinated pigs displayed vaccine-associated enhanced respiratory disease (VAERD) following heterologous challenge, characterized by enhanced macroscopic lung lesions. This study demonstrates that a single intranasal vaccination with an Ad5-HA construct can provide complete protection from homologous challenge and partial protection from heterologous challenge, as opposed to VAERD, which can occur with adjuvanted WIV vaccines.     

Virus-Like Particle Vaccine Confers Protection against a Lethal Newcastle Disease Virus Challenge in Chickens and Allows a Strategy of Differentiating Infected from Vaccinated Animals

In this study, we developed Newcastle disease virus (NDV) virus-like particles (VLPs) expressing NDV fusion (F) protein along with influenza virus matrix 1 (M1) protein using the insect cell expression system. Specific-pathogen-free chickens were immunized with oil emulsion NDV VLP vaccines containing increasing dosages of VLPs (0.4, 2, 10, or 50 μg of VLPs/0.5-ml dose). Three weeks after immunization, the immunogenicity of the NDV VLP vaccines was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit, and a lethal challenge using a highly virulent NDV strain was performed to evaluate the protective efficacy of the NDV VLP vaccines. NDV VLP vaccines elicited anti-NDV antibodies and provided protection against a lethal challenge in a dose-dependent manner. Although the VLP vaccines containing 0.4 and 2 μg of VLPs failed to achieve high levels of protection, a single immunization with NDV VLP vaccine containing 10 or 50 μg could fully protect chickens from a lethal challenge and greatly reduced challenge virus shedding. Furthermore, we could easily differentiate infected from vaccinated animals (DIVA) using the hemagglutination inhibition (HI) test. These results strongly suggest that utilization of NDV VLP vaccine in poultry species may be a promising strategy for the better control of NDV.