Profiling of hMPV F-specific antibodies isolated from human memory B cells.

Human metapneumovirus (hMPV) belongs to the Pneumoviridae family and is closely related to respiratory syncytial virus (RSV). The surface fusion (F) glycoprotein mediates viral fusion and is the primary target of neutralizing antibodies against hMPV. Here we report 113 hMPV-F specific monoclonal antibodies (mAbs) isolated from memory B cells of human donors. We characterize the antibodies' germline usage, epitopes, neutralization potencies, and binding specificities. We find that unlike RSV-F specific mAbs, antibody responses to hMPV F are less dominant against the apex of the antigen, and the majority of the potent neutralizing mAbs recognize epitopes on the side of hMPV F. Furthermore, neutralizing epitopes that differ from previously defined antigenic sites on RSV F are identified, and multiple binding modes of site V and II mAbs are discovered. Interestingly, mAbs that bind preferentially to the unprocessed prefusion F show poor neutralization potency. These results elucidate the immune recognition of hMPV infection and provide novel insights for future hMPV antibody and vaccine development.

Immune response and reactogenicity of an unadjuvanted intradermally delivered human papillomavirus vaccine using a first generation Nanopatch™ in rhesus macaques: An exploratory, pre-clinical feasibility assessment.

The human papillomavirus (HPV) 9-valent, recombinant vaccine (Gardasil™9) helps protect young adults (males and females) against anogenital cancers and genital warts caused by certain HPV genotypes (ref. Gardasil™9 insert). This vaccine is administered intramuscularly (IM). The aim of this study was to determine preclinically whether intradermal (ID) vaccination with an unadjuvanted 9-valent recombinant HPV vaccine using a first-generation ID delivery device, the Nanopatch™, could enhance vaccine immunogenicity compared with the traditional ID route (Mantoux technique). IM injection of HPV VLPs formulated with Merck & Co., Inc., Kenilworth, NJ, USA Alum Adjuvant (MAA) were included in the rhesus study for comparison. The Nanopatch™ prototype contains a high-density array comprised of 10,000 microprojections/cm2, each 250 µm long. It was hypothesized the higher density array with shallower ID delivery may be superior to the Mantoux technique. To test this hypothesis, HPV VLPs without adjuvant were coated on the Nanopatch™, stability of the Nanopatch™ with unadjuvanted HPV VLPs were evaluated under accelerated conditions, skin delivery was verified using radiolabelled VLPs or FluoSpheres®, and the immune response and skin site reaction with the Nanopatch™ was evaluated in rhesus macaques. The immune response induced by Nanopatch™ administration, measured as HPV-specific binding antibodies, was similar to that induced using the Mantoux technique. It was also observed that a lower dose of unadjuvanted HPV VLPs delivered with the first-generation Nanopatch™ and applicator or Mantoux technique resulted in an immune response that was significantly lower compared to a higher-dose of alum adjuvanted HPV VLPs delivered IM in rhesus macaques. The study also indicated unadjuvanted HPV VLPs could be delivered with the first-generation Nanopatch™ and applicator to the skin in 15 s with a transfer efficiency of approximately 20%. This study is the first demonstration of patch administration in non-human primates with a vaccine composed of HPV VLPs.

Antibody responses to crucial functional epitopes as a novel approach to assess immunogenicity of vaccine adjuvants.

We are interested in developing a vaccine that prevents genital herpes. Adjuvants have a major impact on vaccine immunogenicity. We compared two adjuvants, an experimental Merck Sharp & Dohme lipid nanoparticle (LNP) adjuvant, LNP-2, with CpG oligonucleotide combined with alum for immunogenicity in mice when administered with herpes simplex virus type 2 (HSV-2) glycoproteins C, D and E (gC2, gD2, gE2). The immunogens are intended to produce neutralizing antibodies to gC2 and gD2, antibodies to gD2 and gE2 that block cell-to-cell spread, and antibodies to gE2 and gC2 that block immune evasion from antibody and complement, respectively. Overall, CpG/alum was better at producing serum and vaginal IgG binding antibodies, neutralizing antibodies, antibodies that block virus spread from cell-to-cell, and antibodies that block immune evasion domains on gC2. We used a novel high throughput biosensor assay to further assess differences in immunogenicity by mapping antibody responses to seven crucial epitopes on gD2 involved in virus entry or cell-to-cell spread. We found striking differences between CpG/alum and LNP-2. Mice immunized with gD2 CpG/alum produced higher titers of antibodies than LNP-2 to six of seven crucial epitopes and produced antibodies to more crucial epitopes than LNP-2. Measuring epitope-specific antibodies helped to define mechanisms by which CpG/alum outperformed LNP-2 and is a valuable technique to compare adjuvants.

Development and application of a higher throughput RSV plaque assay by immunofluorescent imaging.

Viral plaque assays are important tools in the development and evaluation of new antiviral drugs or vaccines in both preclinical and clinical research. While plaque assays are the standard tools to measure infectious virus, the methodology is time-consuming and requires experience in recognizing plaques. The assays are also prone to variation among analysts due to plaque recognition and manual counting errors. Here we describe the development of two simplified plaque assays for measuring RSV virus titers and anti-RSV antibody neutralization titers using 96 well plate formats. First, we evaluated multiple parameters to build up a quantitative plaque assay to measure infectious RSV. We then optimized the assay conditions to assess the fundamental changes from the traditional plaque assay, which were elimination of overnight pre-seeding host cells and addition of a centrifugation step after viral infection of the cells. We designed DoE to refine four key parameters within one experiment for host cell density, host cell volume, viral inoculum volume, host cell and viral mixture incubation time to make this assay more robust. We have also adapted these conditions into a second assay, which was an automated plaque reduction neutralization assay (PRNT) to determine neutralization titers of anti-RSV antibodies. Both assays utilize immune fluorescence staining to detect viral plaques. The images of the immuno-stained wells are captured by the PerkinElmer EnSight instrument and show clear visualization of plaques harvesting on day 3. Software algorithm was specifically designed for automatic counting of these fluorescent "objects". The quantitative plaque assay provided titers of RSV similar to those obtained from the traditional plaque assay. The method has been successfully utilized to screen multiple vaccine candidates in viral shedding efficacy studies. The automated PRNT assay provided antibody neutralizing titers that matched with published data. This automated 96 well plaque assay has made it possible to screen RSV samples in a higher throughput manner, and can be extended to other infectious organisms that form plaques for vaccine or drug evaluation.

Design and characterization of a fusion glycoprotein vaccine for Respiratory Syncytial Virus with improved stability.

Respiratory Syncytial Virus (RSV) infection is the leading cause of lower respiratory tract infection in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. The infectious RSV particle is decorated with a type I viral fusion (F) glycoprotein that structurally rearranges from a metastable prefusion form to a highly stable postfusion form. In people naturally infected with RSV, the neutralizing antibodies primarily recognize the prefusion conformation. Therefore, engineered RSV F protein stabilized in its prefusion conformation has been an attractive strategy for developing RSV F vaccine antigens. Long-term stability at 4 °C or higher is a desirable attribute for a RSV F subunit vaccine antigen. We have previously shown that a prefusion stabilized RSV F construct, DS-Cav1, undergoes conformational changes and forms intermediate structures upon long-term storage at 4 °C. Structure-based design was performed to improve the stability of the RSV F subunit vaccine. We identified additional mutations that further stabilize RSV F protein in its prefusion conformation by using binding to a previously described antigenic site I antibody 4D7 as the screening tool. In addition, we designed and identified variants with increased expression levels, which is another desirable attribute for a subunit vaccine. Our data suggested that an RSV F variant F111 is properly folded, and has improved heat stability as well as stability upon long-term storage at 4 °C. A mouse immunogenicity study demonstrated that no compromise in immunogenicity (both binding and neutralizing antibody levels) was observed with the introduction of these additional mutations.

Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein.

Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7.

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates.

Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.

Immunogenicity of a reduced-dose whole killed rabies vaccine is significantly enhanced by ISCOMATRIX™ adjuvant, Merck amorphous aluminum hydroxylphosphate sulfate (MAA) or a synthetic TLR9 agonist in rhesus macaques.

There is a need for novel rabies vaccines suitable for short course, pre- and post-exposure prophylactic regimens which require reduced doses of antigen to address the current worldwide supply issue. We evaluated in rhesus macaques the immunogenicity of a quarter-dose of a standard rabies vaccine formulated with Merck's amorphous aluminum hydroxylphosphate sulfate adjuvant, the saponin-based ISCOMATRIX™ adjuvant, or a synthetic TLR9 agonist. All adjuvants significantly increased the magnitude and durability of the humoral immune response as measured by rapid fluorescent focus inhibition test (RFFIT). Several three-dose vaccine regimens resulted in adequate neutralizing antibody of ≥ 0.5 IU/ml earlier than the critical day seven post the first dose. Rabies vaccine with ISCOMATRIX™ adjuvant given at days 0 and 3 resulted in neutralizing antibody titers which developed faster and were up to one log10 higher compared to WHO-recommended intramuscular and intradermal regimens and furthermore, passive administration of human rabies immunoglobulin did not interfere with immunogenicity of this reduced dose, short course vaccine regimen. Adjuvantation of whole-killed rabies vaccine for intramuscular injection may therefore be a viable alternative to intradermal application of non-adjuvanted vaccine for both pre- and post-exposure regimens.

A trivalent recombinant Ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous SIV challenge.

It has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eight Indian rhesus macaques with a trivalent replication-incompetent adenovirus serotype 5 vaccine expressing SIV Gag, Pol, and Nef using a regimen similar to that employed in the Step trial. We detected broad vaccine-induced CD8(+) (2-7 pool-specific responses) and CD4(+) (5-19 pool-specific responses) T-cell responses in IFN-γ ELISPOT assays at one week post-boost using fresh PBMC. However, using cryopreserved cells at one and four weeks post-boost we observed a reduction in both the number and magnitude of most vaccine-induced responses. This demonstrates that the time points and conditions chosen to perform immune assays may influence the observed breadth and frequency of vaccine-induced T-cell responses. To evaluate protective efficacy, we challenged the immunized macaques, along with naïve controls, with repeated, limiting doses of the heterologous swarm isolate SIVsmE660. Vaccination did not significantly affect acquisition or control of virus replication in vaccinees compared to naïve controls. Post-infection we observed an average of only two anamnestic CD8(+) T-cell responses per animal, which may not have been sufficiently broad to control heterologous virus replication. While the trivalent vaccine regimen induced relatively broad T-cell responses in rhesus macaques, it failed to protect against infection or control viral replication. Our results are consistent with those observed in the Step trial and indicate that SIV immunization and challenge studies in macaque models of HIV infection can be informative in assessing pre-clinical HIV vaccines.

Low-dose penile SIVmac251 exposure of rhesus macaques infected with adenovirus type 5 (Ad5) and then immunized with a replication-defective Ad5-based SIV gag/pol/nef vaccine recapitulates the results of the phase IIb step trial of a similar HIV-1 vaccine.

The Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8(+) T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (10(3) 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.

The development of recombinant subunit envelope-based vaccines to protect against dengue virus induced disease.

Challenges associated with the interference observed between the dengue virus components within early tetravalent live-attenuated vaccines led many groups to explore the development of recombinant subunit based vaccines. Initial efforts in the field were hampered by low yields and/or improper folding, but the use of the Drosophila S2 cell expression system provided a mechanism to overcome these limitations. The truncated dengue envelope proteins (DEN-80E) for all four dengue virus types are expressed in the S2 system at high levels and have been shown to maintain native-like conformation. The DEN-80E proteins are potent immunogens when formulated with a variety of adjuvants, inducing high titer virus neutralizing antibody responses and demonstrating protection in both mouse and non-human primate models. Tetravalent vaccine formulations have shown no evidence of immune interference between the four DEN-80E antigens in preclinical models. Based on the promising preclinical data, the recombinant DEN-80E proteins have now advanced into clinical studies. An overview of the relevant preclinical data for these recombinant proteins is presented in this review.

Comparison of T cell immune responses induced by vectored HIV vaccines in non-human primates and humans.

Following the disappointing outcome of the phase IIb test-of-concept step study in which Merck's adenovirus type 5 (Ad5) HIV-1 clade B gag/pol/nef vaccine failed to demonstrate efficacy in HIV high-risk individuals, an extensive review of the trial and preclinical studies which supported the trial is ongoing. One point of interest is how well preclinical nonhuman primate immunogenicity studies predicted what was observed in humans. Here we compare the HIV-1-specific cellular immune responses elicited in nonhuman primates and human clinical trial subjects to several HIV-1 vaccine candidates. We find that although rhesus macaques are immunologically more responsive to vaccination than humans, the hierarchy in potency of single-modality prime-boost regimens using several vector approaches (adenovirus, DNA, and pox vectors) was well predicted. Vaccine approaches using complex formulations such as novel adjuvants (DNA+CRL1005) or mixed-modality prime-boost (DNA/Ad5; Ad5/ALVAC) did not correlate as well between rhesus macaques and humans. Although the immunogenicity of the vaccines and vaccine regimens evaluated were not all accurately predicted, testing in rhesus macaques generally offers an indispensable tool for ranking the immunological potential of HIV-1 vaccine candidates.

Comparative analysis of immune responses induced by vaccination with SIV antigens by recombinant Ad5 vector or plasmid DNA in rhesus macaques.

DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFNgamma responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8(+) T cells, and increased polyfunctionality of both CD4(+) and CD8(+) T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.

Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.

Robust, vaccine-induced CD8(+) T lymphocyte response against an out-of-frame epitope.

Rational vaccines designed to engender T cell responses require intimate knowledge of how epitopes are generated and presented. Recently, we vaccinated 8 Mamu-A*02(+) rhesus macaques with every SIV protein except Envelope (Env). Surprisingly, one of the strongest T cell responses engendered was against the Env protein, the Mamu-A*02-restricted epitope, Env(788-795)RY8. In this paper, we show that translation from an alternate reading frame of both the Rev-encoding DNA plasmid and the rAd5 vector engendered Env(788-795)RY8-specific CD8(+) T cells of greater magnitude than "normal" SIV infection. Our data demonstrate both that the pathway from vaccination to immune response is not well understood and that products of alternate reading frames may be rich and untapped sources of T cell epitopes.

Safety and immunogenicity of the Merck adenovirus serotype 5 (MRKAd5) and MRKAd6 human immunodeficiency virus type 1 trigene vaccines alone and in combination in healthy adults.

Preexisting immunity to adenovirus serotype 5 (Ad5) diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specific neutralizing antibodies and enhance immune responses in populations with a high prevalence of Ad5 immunity. To test this hypothesis, healthy human immunodeficiency virus (HIV)-seronegative adults were enrolled in a blinded, randomized, dose-escalating, placebo-controlled study. In part A, subjects with baseline Ad6 titers of < or = 18 received the Merck Ad6 (MRKAd6) HIV type 1 (HIV-1) trigene vaccine at weeks 0, 4, and 26. In part B, subjects stratified by Ad5 titers (< or = 200 or >200) and Ad6 titers (< or = 18 or >18) received the MRKAd5-plus-MRKAd6 (MRKAd5+6) HIV-1 trigene vaccine at weeks 0, 4, and 26. Immunogenicity was assessed by an enzyme-linked immunospot (ELISPOT) assay at week 30. No serious adverse events occurred. MRKAd6 trigene vaccine recipients responded more often to Nef than to Gag or Pol. In part A, ELISPOT response rates to > or = 2 vaccine antigens were 14%, 63%, and 71% at 10(9), 10(10), and 10(11) viral genomes (vg)/dose, respectively. All responders had positive Nef-specific ELISPOT results. In part B, Nef-ELISPOT response rates at 10(10) vg/dose of the MRKAd5+6 trigene vaccine were 50% in the low-Ad5/low-Ad6 stratum (n = 8), 78% in the low-Ad5/high-Ad6 stratum (n = 9), 75% in the high-Ad5/low-Ad6 stratum (n = 8), and 44% in the high-Ad5/high-Ad6 stratum (n = 9). The MRKAd6 and MRKAd5+6 trigene vaccines elicited dose-dependent responses predominantly to Nef and were generally well tolerated, indicating that Ad6 should be considered a candidate vector for future vaccines. Although small sample sizes limit the conclusions that can be drawn from this exploratory study, combining two Ad vectors may be a useful vaccine strategy for circumventing isolated immunity to a single Ad serotype.

Vaccine-induced cellular responses control simian immunodeficiency virus replication after heterologous challenge.

All human immunodeficiency virus (HIV) vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly reactive neutralizing antibodies. To explore the extent to which vaccine-induced cellular immune responses, in the absence of neutralizing antibodies, can control replication of a heterologous, mucosal viral challenge, we vaccinated eight macaques with a DNA/Ad5 regimen expressing all of the proteins of SIVmac239 except Env. Vaccinees mounted high-frequency T-cell responses against 11 to 34 epitopes. We challenged the vaccinees and eight naïve animals with the heterologous biological isolate SIVsmE660, using a regimen intended to mimic typical HIV exposures resulting in infection. Viral loads in the vaccinees were significantly less at both the peak (1.9-log reduction; P < 0.03) and at the set point (2.6-log reduction; P < 0.006) than those in control naïve animals. Five of eight vaccinated macaques controlled acute peak viral replication to less than 80,000 viral RNA (vRNA) copy eq/ml and to less than 100 vRNA copy eq/ml in the chronic phase. Our results demonstrate that broad vaccine-induced cellular immune responses can effectively control replication of a pathogenic, heterologous AIDS virus, suggesting that T-cell-based vaccines may have greater potential than previously appreciated.

Serotype specificity of adenovirus purification using anion-exchange chromatography.

Recombinant adenoviruses continue to be a leading vector choice for gene transfer applications, with growing interest in the use of less prevalent serotypes, and of chimeras. As a result, the development of scaleable purification processes for alternative serotypes is needed. Anion-exchange chromatography is routinely used for scaleable adenovirus type 5 purification; however, retention varies for other serotypes because of differences in the exposed capsid proteins. Understanding how the viral surface influences retention behavior can provide a rational basis for chromatography development and optimization. In this work, chimeric vectors were used to show that the hexon protein is responsible for retention differences in anion-exchange chromatography. Next, the relative retention of eight serotypes from three subgroups was studied. Although all serotypes bound to the anion-exchange resin, the sodium chloride required to elute the virus varied over a 2- fold range, from 270 to 490 mM. Retention was accurately correlated to the electrostatic properties of the hexon protein, with an average error in sodium chloride concentration required to elute of only 14 mM. This correlation enables preparative chromatography gradients for alternative serotypes to be established with minimal effort.

Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag.

The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(-) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(-) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.

Heterologous human immunodeficiency virus type 1 priming-boosting immunization strategies involving replication-defective adenovirus and poxvirus vaccine vectors.

We compared the human immunodeficiency virus type 1 (HIV-1)-specific cellular immune responses elicited in nonhuman primates by HIV-1 gag-expressing replication-defective adenovirus serotype 5 (Ad5) or poxvirus vectors, used either alone or in combination with each other. The responses arising from a heterologous Ad5 priming-poxvirus boosting regimen were significantly greater than those elicited by homologous regimens with the individual vectors or by a heterologous poxvirus priming-Ad5 boosting regimen. The heterologous Ad5 priming-poxvirus boosting approach may have potential utility in humans as a means of inducing high levels of cellular immunity.