Induction of neutralizing antibodies to T-cell line-adapted and primary human immunodeficiency virus type 1 isolates with a prime-boost vaccine regimen in chimpanzees.

Five chimpanzees were immunized by administration of one or more intranasal priming doses of one to three recombinant adenoviruses containing a gp160 insert from human immunodeficiency virus type 1 (HIV-1) MN (HIV-1MN) followed by one or more boosts of recombinant HIV-1SF2 gp120 delivered intramuscularly with MF59 adjuvant. This regimen resulted in humoral immune responses in three of five animals. Humoral responses included immunochemically active anti-H1V-1 antibodies (Abs) directed to recombinant gp120 and neutralizing Abs reactive with T-cell-line-adapted HIV-1MN and HIV-1SF2. In addition, neutralizing activity was detected to the two homologous primary isolates and to two of three heterologous primary isolates which, like the immunizing strains, can use CXCR4 as a coreceptor for infection. The three animals with detectable neutralizing Abs and a fourth exhibiting the best cytotoxic T-lymphocyte response were protected from a low-dose intravenous challenge with a cell-free HIV-1SF2 primary isolate administered 4 weeks after the last boost. Animals were rested for 46 weeks and then rechallenged, without a boost, with an eightfold-higher challenge dose of HIV-1SF2. The three animals with persistent neutralizing Abs were again protected. These data show that a strong, long-lived protective Ab response can be induced with a prime-boost regimen in chimpanzees. The data suggest that in chimpanzees, the presence of neutralizing Abs correlates with protection for animals challenged intravenously with a high dose of a homologous strain of HIV-1, and they demonstrate for the first time the induction of neutralizing Abs to homologous and heterologous primary isolates.

Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Immunogenicity of recombinant adenovirus-human immunodeficiency virus vaccines in chimpanzees following intranasal administration.

Recombinant adenovirus (Ad)-human immunodeficiency virus (HIV) vaccines expressing HIVIIIB Env and Gag proteins were evaluated for immunogenicity in chimpanzees following intranasal administration. When Ad7-, Ad4-, and Ad5-vectored vaccines were administered sequentially at 0, 24, and 52 weeks, respectively, to three chimpanzees, the inoculations resulted in limited virus replication in the nasopharynx, but extensive Ad-HIV replication occurred in the intestine. High-titered IgG serum antibody responses to Env and Gag that were nonneutralizing were induced following booster administration of Ad4-HIV recombinant viruses. Following the Ad5-HIV booster, low levels of neutralizing antibodies as well as V3 loop antibodies were induced in all three chimpanzees that persisted for several months. Administration of a gp160 subunit vaccine (baculovirus derived) in SAF-m 24 weeks later boosted broadly neutralizing serum antibodies that peaked within 1 month of the injection. Two additional subunit boosters 19 and 37 weeks later were progressively less effective at stimulating serum neutralizing antibody responses. Substantial local immune responses were induced in nasal, vaginal, and salivary secretions following the third Ad-HIV intranasal immunization. These responses were further boosted with the gp160 subunit vaccine, which also stimulated production of rectal antibodies. The predominant responses in all secretions tested were of the IgG isotype, although some IgA responses were also detected. Strong blastogenic responses to HIV recombinant Env and Gag proteins were induced after each immunization.

Adenovirus vectored vaccines.

Human recombinant adenoviruses (Ad) have been employed to develop experimental vaccines against a number of infectious agents. Ad-vectored vaccines express recombinant proteins, including any post-translational modifications, into functioning replicas of the native proteins capable of eliciting neutralizing antibodies in both abortive and permissive animal models. Human Ad types 4, 5, and 7 were used to construct recombinant viruses that express the respiratory syncytial virus F or G glycoproteins, the hepatitis B surface antigen, and the HIV env or gag genes. The recombinant Ad-HIV viruses are of particular interest and have been examined for their immunogenicity in dogs and chimpanzees. Dogs were immunized intratracheally with Ad-env recombinants (10(9) pfu/dog). Excellent humoral anti-HIV responses, including neutralizing antibodies, were detected in the sera following booster immunization (12-18 weeks after primary immunization) with a second Ad-env recombinant made in a different Ad serotype (heterotypic booster). Chimpanzees were immunized in two ways, orally with lyophilized virus (10(9) to 10(10) pfu/virus) in enteric-coated capsules or intranasally (10(7) pfu/virus). Intranasal immunization was superior to oral immunization with respect to replication of recombinant viruses as well as induction of anti-Ad and anti-HIV antibodies. Administration by both routes resulted in stimulation of cellular immune responses, as measured by antigen proliferation assays. Anti-HIV antibodies were detected in chimpanzee secretions (salivary, nasal, rectal, vaginal) taken from animals following intranasal immunization with a heterotypic recombinant. Intranasal administration effectively primed chimpanzees to produce high-titred (320-640) serum neutralizing antibodies to HIV following boosting with a baculovirus-derived env (gp160) subunit vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees.

Recombinant human adenovirus (Ad) type 4-, 5-, and 7-vectored vaccines expressing either the HIV env or gag-protease genes were tested for immunogenicity in three chimpanzees. The first phase of the vaccination protocol consisted of a primary and two booster immunizations with Ad-HIVs by the oral route of administration, followed by a single booster immunization with Gag and/or Env subunit vaccines. The second phase of the vaccination protocol consisted of intranasal administration of Ad-HIVs previously administered by the oral route. Following the first phase adenovirus was shed into stools for only 1-7 days and modest type-specific anti-adenovirus neutralizing antibody titers were induced. Strong anti-Env binding antibody responses were detected in all three animals following the second oral booster immunization. One chimpanzee responded with a low-titered type-specific neutralizing antibody response to HIV. Cell-mediated immune responses to Env were not detected after the primary vaccination, but were detected following all booster immunizations. Administration of the Gag subunit vaccine boosted both humoral and cell-mediated immune responses to Gag antigens. In contrast, the Env subunit vaccine boosted cellular but not humoral immune responses. In the second phase of the vaccination protocol, both virus shedding and anti-adenovirus responses were enhanced. All three chimpanzees responded to the intranasal administration of Ad7-HIVs with boosted anti-HIV serum responses, including low-titered type-specific neutralizing antibodies, elicited anti-HIV antibodies at secretory sites, and stimulated cell-mediated immune responses to both Gag and Env antigens.

Adenovirus-human immunodeficiency virus (HIV) envelope recombinant vaccines elicit high-titered HIV-neutralizing antibodies in the dog model.

Recombinant human adenoviruses (Ads) (types 4, 5, and 7) expressing the HIV-1 envelope membrane glycoprotein (gp160) were tested for immunogenicity in the dog. Administration of recombinant Ad7-env by intratracheal inoculation resulted in a low serum antibody response to gp160, which developed over several weeks. A strong neutralizing antibody response to the Ad7 vector developed within 1 week of infection. A subsequent booster inoculation 12 weeks later with the heterotypic Ad4-env recombinant virus resulted in significantly enhanced humoral responses directed at the envelope antigen, as measured by both ELISA and Western blot analysis as well as high-titer type-specific neutralizing antibodies, with some animals achieving neutralization titers approaching 1000. Recombinant HIV envelope glycoprotein derived from Ad-HIV-infected cell cultures was used as a subunit booster injection for dogs that had previously received sequential immunizations with heterotypic recombinant Ads. Significant immune responses against the envelope developed as measured by ELISA, Western blot analysis, and neutralization assays. These data indicate that live recombinant Ad-HIV vaccines are capable of inducing high-titer type-specific neutralizing antibodies to gp160 in vivo. Recombinant HIV envelope glycoprotein subunit vaccines, prepared from Ad-env-infected cells, are capable of boosting these responses.

Adenovirus vectors for gene expression.

Adenoviruses possess a combination of features that make them highly suitable as vectors for expression of heterologous genes. Non-conditional and non-defective adeno-vectors have been constructed to obtain high level expression of a number of foreign genes and some of them have been shown in animal models to exhibit excellent promise as vaccine candidates.

High level expression of the envelope glycoproteins of the human immunodeficiency virus type I in presence of rev gene using helper-independent adenovirus type 7 recombinants.

The effect of rev (art/trs) gene on the level of HIV-1 envelope (env) expression using recombinant adenovirus was investigated. Recombinant adenoviruses expressing either the envelope or the rev gene of the human immunodeficiency virus type 1 (HIV-1) were constructed by inserting the gene into an expression cassette. The expression cassette contained the adenovirus type 7 major late promoter, followed by leader 1 of the adenovirus tripartite leader and a portion of intron between leaders 1 and 2, leaders 2 and 3, and a hexon polyadenylation signal. The cassette was then inserted at the terminal region between the E4 and ITR regions of the adenovirus 7 genome with a concomitant E3 region deletion (80-87 m.u.). A549 cells infected with the recombinant virus containing the env gene produced the envelope glycoproteins gp160, gp120, and gp41. HIV-1 envelope gene expression was greatly enhanced (20- to 50-fold) in the cells that were simultaneously infected with the recombinant adenovirus containing the rev gene as measured by ELISA and Western blotting. Interestingly, this effect was observed despite the lack of the 5' down splice site for rev and seems to be post-transcriptional. Another recombinant adenovirus which contains both the rev and the env genes was constructed by inserting the rev gene in the deleted E3 region and the env gene in the terminal cassette. This double recombinant virus expressed high levels of env antigen in A549 cells similar to those attained upon co-infection with two separate recombinant viruses containing the rev or env gene. Furthermore, the rev gene nucleotide sequence could be altered without altering the amino acid sequence and its sequences truncated by 17 amino acids from the C-terminus had no effect of rev function.

Adenovirus vaccine strains genetically engineered to express HIV-1 or HBV antigens for use as live recombinant vaccines.

Types 4 and 7 adenovirus are currently used as live, oral vaccines for the prevention of adenovirus respiratory disease in military recruits. These vaccine strains have been genetically engineered in order to express HIV-1 or HBV antigens in infected cells. A dog model was developed to evaluate the immunogenicity of these recombinant vaccines. Dogs inoculated with live adenovirus-HBV recombinant vaccine produced antibody against hepatitis B surface antigen.

Antiviral effect of lymphokine-activated killer cells: chemotaxis and homing to sites of virus infection.

Lymphokine-activated killer (LAK) cells generated from C57BL/6 mouse spleen cells cultured with interleukin-2 are effective prophylactically against virus infection when inoculated at the site of virus injection. To predict the therapeutic efficacy of LAK cells, we determined whether LAK cells would home to sites of virus infection. In vitro, LAK cells responded chemotactically to cell-free peritoneal exudate fluids collected from virus-infected mice and to preparations of purified beta interferon. In vivo, radiolabeled LAK cells injected intravenously accumulated in the peritoneal cavities of intraperitoneally infected mice in amounts three to eight times greater than in uninfected mice. This ability to respond to chemotactic agents and migrate into sites of virus infection may make LAK cells useful as antiviral therapeutic agents.

Immunogenicity and efficacy testing in chimpanzees of an oral hepatitis B vaccine based on live recombinant adenovirus.

As a major cause of acute and chronic liver disease as well as hepatocellular carcinoma, hepatitis B virus (HBV) continues to pose significant health problems world-wide. Recombinant hepatitis B vaccines based on adenovirus vectors have been developed to address global needs for effective control of hepatitis B infection. Although considerable progress has been made in the construction of recombinant adenoviruses that express large amounts of HBV gene products, preclinical immunogenicity and efficacy testing of candidate vaccines has remained difficult due to the lack of a suitable animal model. We demonstrate here that chimpanzees are susceptible to enteric infection by human adenoviruses type 7 (Ad7) and type 4 (Ad4) following oral administration of live virus. Moreover, after sequential oral immunization with Ad7- and Ad4-vectored vaccines containing the hepatitis B surface antigen (HBsAg) gene, significant antibody responses to HBsAg (anti-HBs) were induced in two chimpanzees. After challenge with heterologous HBV, one chimpanzee was protected from acute hepatitis and the other chimpanzee experienced modified HBV-induced disease. These data demonstrate the feasibility of using orally administered recombinant adenoviruses as a general approach to vaccination.

Blastogenesis of large granular lymphocytes in nonlymphoid organs.

High numbers of large granular lymphocytes (LGL) accumulate in the livers and peritoneal cavities of mice during the course of viral infection. Accumulation of natural killer (NK) cells at day 3 postinfection (p.i.) was shown to be radiation-sensitive, implying that proliferation was required for this response. Accumulation occurred in splenectomized mice, indicating that the spleen, known to be an organ for mature NK cell proliferation, was not the major source for liver and peritoneal NK/LGL. Significant percentages (greater than 25%) of the LGL found in the liver and peritoneal cavity following viral infection or interferon induction with poly-inosinic:poly-cytidylic acid were defined morphologically as blasts (large cells with prominent nucleoli and intensely basophilic cytoplasms containing azurophilic granules). Most blast LGL at day 3 p.i. were sensitive to administration of anti-asialo GM1 serum in vivo, were Lyt-2-, and were enriched in populations that lysed NK cell-sensitive targets in vitro, indicating that these were NK/LGL. At day 3 p.i., leukocytes from the liver and peritoneal cavity incorporated 3H-thymidine and bound to and killed NK cell-sensitive targets in single-cell cytotoxicity assays. These data suggest that NK/LGL undergo at least one round of division in the liver and peritoneal cavity during viral infection. In contrast, blast LGL at day 7 p.i. were resistant to in vivo treatments with anti-asialo GM1 serum, were Lyt-2+, and were enriched in populations of cells that killed virus-infected histocompatible targets, indicating that they were cytotoxic T lymphocytes (CTL). These results suggest that both NK/LGL and CTL/LGL are capable of blastogenesis and presumed proliferation at sites of virus infection, providing a means for the in situ augmentation of a host's cell-mediated antiviral defenses.

Chemotactic effect of human recombinant interleukin 2 on mouse activated large granular lymphocytes.

Human recombinant interleukin 2 (hrIL-2) was demonstrated in vitro to be chemotactic for mouse large granular lymphocytes (LGL) activated in vivo by virus infection. Peritoneal exudate cells harvested from virus-infected mice were used as a source of LGL. LGL collected from mouse hepatitis virus-infected mice at 3 days postinfection were a source for NK 1.1 positive natural killer (NK)/LGL. LGL collected from mice treated with antiserum to gangliotetraosylceramide and infected with lymphocytic choriomeningitis virus for 7 days were used as a source for Lyt-2 positive cytotoxic T lymphocytes (CTL)/LGL. Both NK/LGL and CTL/LGL responded chemotactically to hrIL-2, purified IFN-beta, and to crude cell-free washout fluids collected from the peritoneal cavity of virus-infected mice. hrIL-2 had chemotactic activity for virus-elicited granular and agranular lymphocytes but did not attract the contaminating macrophages, in contrast to IFN-beta, which displayed chemotactic activity for virus-elicited granular and agranular lymphocytes as well as macrophages. The migration to hrIL-2 was inhibited by a monoclonal antibody (7D4) to the IL-2 receptor, but treatment with 7D4 did not affect migration in response to IFN-beta. Microscopic examination of Wright's-Giemsa-stained migrated NK/LGL and CTL/LGL revealed that the majority of migrated LGL in either LGL population had a blast cell morphology (enlarged cells with rich basophilic cytoplasm). The frequency of cells bearing the LGL morphology within the virus-elicited nonadherent peritoneal exudate cell population was on incubation in vitro, stabilized by either hrIL-2 or IFN-beta. These data suggest that another important immunomodulating function of IL-2 may be to attract activated NK/LGL and CTL/LGL to sites of inflammation.

Accumulation and chemotaxis of natural killer/large granular lymphocytes at sites of virus replication.

A model for monitoring the accumulation of natural killer cell/large granular lymphocytes (NK/LGL) at a site of virus replication was studied by using mice infected i.p. with either lymphocytic choriomeningitis virus (LCMV), murine cytomegalovirus (MCMV), mouse hepatitis virus (MHV), Pichinde virus, or vaccinia virus. An i.p. but not i.v. infection resulted in a localized increase in NK/LGL cell number (a fourfold to greater than 20-fold increase) and augmentation (a 10- to 20-fold increase) of NK cell activity associated with virus-induced peritoneal exudate cell (PEC) populations. An increase in NK/LGL cell number was detected as early as 12 hr postinfection (p.i.) and peaked at 3 days p.i. with MHV. The initial LGL recruited into the peritoneal cavity at 1 to 3 days p.i. were nonadherent to plastic and were demonstrated to have an NK cell phenotype: asialo GM1+, Thy-1.2 +/-, Lyt-2.2-, and J11d-. The peak number of LGL appeared at 7 days after infection with the NK cell-resistant virus, LCMV. This LGL population had been previously demonstrated to contain cytotoxic T lymphocyte/LGL (CTL/LGL) as well as NK/LGL. During an MHV infection the number of LGL decreased between days 3 and 7 p.i., suggesting that the second wave of CTL/LGL was absent. These findings may explain the absence of a good MHV-CTL model. Virus-induced, activated NK/LGL responded to chemotactic signals by migrating in a unidirectional manner across two 5-microns pore size polycarbonate filters during 7 hr in vitro chemotaxis assays. Wash-out fluid obtained from the peritoneal cavity contained chemotactic activity for NK/LGL as well as for other cell types. We conclude that production and/or release of chemotactic factors at sites of virus replication are at least partially responsible for the accumulation of NK/LGL at these sites.

Generation of large granular T lymphocytes in vivo during viral infection.

Cytolytic lymphocytes were isolated from the spleens of lymphocytic choriomeningitis virus (LCMV)-infected mice and were characterized in regards to function, cell size, antigen phenotype, and cell morphology. Only 2% of the Lyt-2+ cells from uninfected mice were large granular lymphocytes (LGL), whereas 21% of the Lyt-2+ cells isolated 7 days postinfection were LGL. The day 7 Lyt-2+ populations contained all of the LCMV-specific, class I histocompatibility antigen-restricted cytotoxic T lymphocyte (CTL) activity, but no natural killer (NK) cell activity. The NK cell activity was consistently recovered in Lyt-2- populations isolated from both control mice and mice on day 7 postinfection. The LGL isolated on day 7 postinfection were concluded to be predominantly T cells and not NK cells because 1) the proportions of LGL in fractionated cell populations 7 days postinfection correlated with levels of CTL-mediated lysis but not NK cell-mediated lysis, 2) they were recovered in the Lyt-2+ population, and 3) antibody to asialo GM1, known to eliminate NK cell-mediated lysis but not T cell-mediated lysis, dramatically reduced NK cell LGL numbers in vivo on day 3 postinfection but only marginally affected LGL numbers on day 7. Virus-induced inflammation elicited a 50-fold increase in LGL numbers in the peritoneum on day 7 postinfection. The peritoneal exudate LGL were also associated with CTL activity and were resistant to treatment with antibody to asialo GM1. These results indicate that in vivo-generated CTL have the morphology of LGL and that the appearance of cytoplasmic granules correlates with the ability of cells to mediate lysis. To focus on cells being stimulated during infections, activated blast cells were separated from small resting cells by centrifugal elutriation. Coincidental with the peak in overall spleen leukocyte cytotoxic activity, the peaks of blast NK cells and CTL were at days 3 and 7 postinfection respectively. More than 50% of the blast lymphocytes isolated on either day 3 or day 7 postinfection were LGL. The CTL activity in the blast populations on day 7 postinfection was mediated by Lyt-2+ cells, and 37 to 64% of these Lyt-2+ blast cells were LGL. Cytolytic NK cell and CTL LGL could not be distinguished by morphology or by cell densities, because they overlapped in low density Percoll gradient fractions. Since this technique has been used to enrich for LGL, these data indicate that heterogeneity in LGL populations may result from the presence of both CTL and NK cell LGL.

Infection of DBA/2 or C3H/HeJ mice by intraperitoneal injection of vaccinia virus elicits activated macrophages, cytolytic and cytostatic for S91-melanoma tumor cells.

Murine peritoneal macrophages harvested 3-4 days after IP injection of vaccinia virus lysed S91-melanoma tumor cells in vitro; enhanced tumoricidal activity was measured with effector macrophages prepared 5-6 days after vaccinia virus infection. Treatment of virus-elicited macrophages prepared from DBA/2 mice with anti-asialo-GM1 antiserum, anti-Thy 1.2 antiserum or anti-Iad antiserum in the presence of complement so that cells sensitized with antibodies were lysed, did not reduce the measured level of tumoricidal activity indicating that macrophages [Ia(-); asialo GM1(-)] and not natural killer cells [asialo GM1(+); Thy 1.2(+/-)] or T-cells [Thy 1.2(+)] were responsible for mediating the lysis of S91-melanoma tumor cells. When incubated with virus-elicited macrophages but not thioglycollate-elicited macrophages, the ability of S91-melanoma tumor cells. to synthesize DNA was completely blocked. The results of these experiments support the view that one aspect of antitumor immunity enhanced during immunotherapy with vaccinia virus is the activation of macrophages which have cytolytic as well as cytostatic effects on melanoma tumor cells.

Vaccinia virus proteins on the plasma membrane of infected cells. III. Infection of peritoneal macrophages.

Primary macrophage cultures were prepared from the peritoneal exudate cell population harvested from mice challenged intraperitoneally with saline, thioglycollate, or vaccinia virus. Vaccinia virus was adsorbed and penetrated into primary macrophages and L-cells with similar kinetics. As evidenced by the expression of some "early" virus-specified proteins, partial uncoating and activation of the virion-associated DNA-dependent RNA polymerase occurred in the infected macrophages. Subsequently, the viral replication cycle in macrophages was aborted; with time after infection, viral DNA and virion proteins initially associated with infected cells could be detected in an acid-soluble form in the medium harvested from infected macrophage cultures. The results suggest that at the time that the final stages of virus uncoating should have occurred, intracellular subviral particles were, instead, degraded in the infected, primary macrophages. Viral DNA synthesis could not be measured in vaccinia virus-infected macrophages, no "late" virus functions were expressed, and progeny virions were not assembled. As measured by the binding of antiviral-antibody-125I-protein A complexes to the surface of vaccinia virus-infected cells, the expression of virus-specified antigens on the surfaces of infected macrophages was significantly reduced and never exceeded that measured at 2 hr after infection on the surfaces of infected L-cells. The expression of virus-specified polypeptides with mol mass of 48-50, 45-46, 36-37, and 25 kDa on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages, rendered the infected macrophages susceptible to lysis by vaccinia virus-specific cytotoxic T-cells.