HIV-1 integration landscape during latent and active infection.

The barrier to curing HIV-1 is thought to reside primarily in CD4(+) T cells containing silent proviruses. To characterize these latently infected cells, we studied the integration profile of HIV-1 in viremic progressors, individuals receiving antiretroviral therapy, and viremic controllers. Clonally expanded T cells represented the majority of all integrations and increased during therapy. However, none of the 75 expanded T cell clones assayed contained intact virus. In contrast, the cells bearing single integration events decreased in frequency over time on therapy, and the surviving cells were enriched for HIV-1 integration in silent regions of the genome. Finally, there was a strong preference for integration into, or in close proximity to, Alu repeats, which were also enriched in local hotspots for integration. The data indicate that dividing clonally expanded T cells contain defective proviruses and that the replication-competent reservoir is primarily found in CD4(+) T cells that remain relatively quiescent.

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection.

Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome.

During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model, which enables exploring the parameter space of the WT and DI competition. We also experimentally examined virus and DI replication kinetics during co-infection, and used these data to infer model parameters. Our model identifies, and our experimental measurements confirm, that the efficiencies of DI genome replication and encapsidation are two most critical parameters determining the outcome of WT replication. However, an equilibrium can be established which enables WT to replicate, albeit to reduced levels.

Polyamine Depletion Inhibits Bunyavirus Infection via Generation of Noninfectious Interfering Virions.

Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies.IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.

Generation of a purely clonal defective interfering influenza virus.

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus-based antivirals.

Identification and characterization of viral defective RNA genomes in influenza B virus.

Influenza B virus (FLUBV) is an important pathogen that infects humans and causes seasonal influenza epidemics. To date, little is known about defective genomes of FLUBV and their roles in viral replication. In this study, by using a next-generation sequencing approach, we analyzed total mRNAs extracted from A549 cells infected with B/Brisbane/60/2008 virus (Victoria lineage), and identified four defective FLUBV genomes with two (PB1∆A and PB1∆B) from the polymerase basic subunit 1 (PB1) segment and the other two (M∆A and M∆B) from the matrix (M) protein-encoding segment. These defective genomes contained significant deletions in the central regions with each having the potential for encoding a novel polypeptide. Significantly, each of the discovered defective RNAs can potently inhibit the replication of B/Yamanashi/166/98 (Yamagata lineage). Furthermore, PB1∆A was able to interfere modestly with influenza A virus (FLUAV) replication. In summary, our study provides important initial insights into FLUBV defective-interfering genomes, which can be further explored to achieve better understanding of the replication, pathogenesis and evolution of FLUBV.

Spatial-Temporal Patterns of Viral Amplification and Interference Initiated by a Single Infected Cell.

UNLABELLED: When viruses infect their host cells, they can make defective virus-like particles along with intact virus. Cells coinfected with virus and defective particles often exhibit interference with virus growth caused by the competition for resources by defective genomes. Recent reports of the coexistence and cotransmission of such defective interfering particles (DIPs) in vivo, across epidemiological length and time scales, suggest a role in viral pathogenesis, but it is not known how DIPs impact infection spread, even under controlled culture conditions. Using fluorescence microscopy, we quantified coinfections of vesicular stomatitis virus (VSV) expressing a fluorescent reporter protein and its DIPs on BHK-21 host cell monolayers. We found that viral gene expression was more delayed, infections spread more slowly, and patterns of spread became more "patchy" with higher DIP inputs to the initial cell. To examine how infection spread might depend on the behavior of the initial coinfected cell, we built a computational model, adapting a cellular automaton (CA) approach to incorporate kinetic data on virus growth for the first time. Specifically, changes in observed patterns of infection spread could be directly linked to previous high-throughput single-cell measures of virus-DIP coinfection. The CA model also provided testable hypotheses on the spatial-temporal distribution of the DIPs, which remain governed by their predator-prey interaction. More generally, this work offers a data-driven computational modeling approach for better understanding of how single infected cells impact the multiround spread of virus infections across cell populations. IMPORTANCE: Defective interfering particles (DIPs) compete with intact virus, depleting host cell resources that are essential for virus growth and infection spread. However, it is not known how such competition, strong or weak, ultimately affects the way in which infections spread and cause disease. In this study, we address this unmet need by developing an integrated experimental-computational approach, which sheds new light on how infections spread. We anticipate that our approach will also be useful in the development of DIPs as therapeutic agents to manage the spread of viral infections.

Assembly of Replication-Incompetent African Horse Sickness Virus Particles: Rational Design of Vaccines for All Serotypes.

UNLABELLED: African horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids. The virion particle is composed of seven proteins organized in three concentric layers, an outer layer made of VP2 and VP5, a middle layer made of VP7, and inner layer made of VP3 that encloses a replicase complex of VP1, VP4, and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious candidate vaccines against all AHSV serotypes, taking into account not only immunogenic and safety properties but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established a highly efficient reverse genetics (RG) system for AHSV serotype 1 (AHSV1) and, subsequently, a VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required the exchange of two to five RNA segments to achieve equivalent titers of particles. All reassortant-defective viruses could be amplified and propagated to high titers in cells complemented with VP6 but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor (IFNAR)-knockout mice. Thus, these defective viruses have the potential to be used for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis, and pathogenesis. IMPORTANCE: African horse sickness virus is transmitted by biting midges and causes African horse sickness in equids, with mortality reaching up to 95% in naive horses. Therefore, the development of efficient vaccines is extremely important due to major economic losses in the equine industry. Through the establishment of a highly efficient RG system, replication-deficient viruses of all nine AHSV serotypes were generated. These defective viruses achieved high titers in a cell line complemented with VP6 but failed to propagate in wild-type mammalian or insect cells. Importantly, these candidate vaccine strains showed strong protective efficacy against AHSV infection in an IFNAR(-/-) mouse model.

Defective interfering influenza virus RNAs: time to reevaluate their clinical potential as broad-spectrum antivirals?

Defective interfering (DI) RNAs are highly deleted forms of the infectious genome that are made by most families of RNA viruses. DI RNAs retain replication and packaging signals, are synthesized preferentially over infectious genomes, and are packaged as DI virus particles which can be transmitted to susceptible cells. Their ability to interfere with the replication of infectious virus in cell culture and their potential as antivirals in the clinic have long been known. However, until now, no realistic formulation has been described. In this review, we consider the early evidence of antiviral activity by DI viruses and, using the example of DI influenza A virus, outline developments that have led to the production of a cloned DI RNA that is highly active in preclinical studies not only against different subtypes of influenza A virus but also against heterologous respiratory viruses. These data suggest the timeliness of reassessing the potential of DI viruses as a novel class of antivirals that may have general applicability.

Quantitative characterization of defective virus emergence by deep sequencing.

UNLABELLED: Populations of RNA viruses can spontaneously produce variants that differ in genome size, sequence, and biological activity. Defective variants that lack essential genes can nevertheless reproduce by coinfecting cells with viable virus, a process that interferes with virus growth. How such defective interfering particles (DIPs) change in abundance and biological activity within a virus population is not known. Here, a prototype RNA virus, vesicular stomatitis virus (VSV), was cultured for three passages on BHK host cells, and passages were subjected to Illumina sequencing. Reads from the initial population, when aligned to the full-length viral sequence (11,161 nucleotides [nt]), distributed uniformly across the genome. However, during passages two plateaus in read counts appeared toward the 5' end of the negative-sense viral genome. Analysis by normalization and a simple sliding-window approach revealed plateau boundaries that suggested the emergence and enrichment of at least two truncated species having medium (∼5,900 nt) and short (∼4,000 nt) genomes. Relative measures of full-length and truncated species based on read counts were validated by quantitative reverse transcription-PCR (qRT-PCR). Limit-of-detection analysis suggests that deep sequencing can be more sensitive than complementary measures for detecting and quantifying defective particles in a population. Further, particle counts from transmission electron microscopy, coupled with infectivity assays, linked the rise in smaller genomes with an increase in truncated particles and interference activity. In summary, variation in deep sequencing coverage simultaneously shows the size, location, and relative level of truncated-genome variants, revealing a level of population heterogeneity that is masked by other measures of viral genomes and particles. IMPORTANCE: We show how deep sequencing can be used to characterize the emergence, diversity, and relative abundance of truncated virus variants in virus populations. Adaptation of this approach to natural isolates may elucidate factors that influence the stability and persistence of virus populations in nature.

Phylodynamic analysis of the emergence and epidemiological impact of transmissible defective dengue viruses.

Intra-host sequence data from RNA viruses have revealed the ubiquity of defective viruses in natural viral populations, sometimes at surprisingly high frequency. Although defective viruses have long been known to laboratory virologists, their relevance in clinical and epidemiological settings has not been established. The discovery of long-term transmission of a defective lineage of dengue virus type 1 (DENV-1) in Myanmar, first seen in 2001, raised important questions about the emergence of transmissible defective viruses and their role in viral epidemiology. By combining phylogenetic analyses and dynamical modeling, we investigate how evolutionary and ecological processes at the intra-host and inter-host scales shaped the emergence and spread of the defective DENV-1 lineage. We show that this lineage of defective viruses emerged between June 1998 and February 2001, and that the defective virus was transmitted primarily through co-transmission with the functional virus to uninfected individuals. We provide evidence that, surprisingly, this co-transmission route has a higher transmission potential than transmission of functional dengue viruses alone. Consequently, we predict that the defective lineage should increase overall incidence of dengue infection, which could account for the historically high dengue incidence reported in Myanmar in 2001-2002. Our results show the unappreciated potential for defective viruses to impact the epidemiology of human pathogens, possibly by modifying the virulence-transmissibility trade-off, or to emerge as circulating infections in their own right. They also demonstrate that interactions between viral variants, such as complementation, can open new pathways to viral emergence.

Potent anti-tumour activity of a novel conditionally replicating adenovirus for melanoma via inhibition of migration and invasion.

BACKGROUND: Conditionally replicating adenoviruses (CRAds) represent a novel class of oncological therapeutic agents. One strategy to ensure tumour targeting is to place the essential viral genes under the control of tumour-specific promoters. Ki67 has been selected as a cancer gene therapy target, as it is expressed in most malignant cells but is barely detectable in most normal cells. This study aimed to investigate the effects of a Ki67 promoter-controlled CRAd (Ki67-ZD55-IL-24) on the proliferation and apoptosis of melanoma cells. METHODS: Melanoma cells were independently treated with Ki67-ZD55-IL-24, ZD55-IL-24, Ki67-ZD55, and ZD55-EGFP. The cytotoxic potential of each treatment was assessed using cell viability measurements. Cell migration and invasion were assayed using cell migration and invasion assays. Apoptosis was assayed using the annexin V-FITC assay, western blotting, reverse transcriptase PCR (RT-PCR), haematoxylin and eosin (H&E) staining, and the TUNEL assay. RESULTS: Our results showed that Ki67-ZD55-IL-24 had significantly enhanced anti-tumour activity as it more effectively induced apoptosis in melanoma cells than the other agents. Ki67-ZD55-IL-24 also caused the most significant inhibition of cell migration and invasion of melanoma cells. Furthermore, apoptosis was induced more effectively in melanoma xenografts in nude mice. CONCLUSIONS: This strategy holds promising potential for the further development of an effective approach to treat malignant melanoma.

Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon.

A method for rapid production of single-round infectious particles (SRIPs) of flavivirus would be useful for viral mutagenesis studies. Here, we established a DNA-based production system for SRIPs of flavivirus. We constructed a Japanese encephalitis virus (JEV) subgenomic replicon plasmid, which lacked the C-prM-E (capsid-pre-membrane-envelope) coding region, under the control of the cytomegalovirus promoter. When the JEV replicon plasmid was transiently co-transfected with a JEV C-prM-E expression plasmid into 293T cells, SRIPs were produced, indicating successful trans-complementation with JEV structural proteins. Equivalent production levels were observed when C and prM-E proteins were provided separately. Furthermore, dengue types 1-4, West Nile, yellow fever or tick-borne encephalitis virus prM-E proteins could be utilized for production of chimaeric flavivirus SRIPs, although the production was less efficient for dengue and yellow fever viruses. These results indicated that our plasmid-based system is suitable for investigating the life cycles of flaviviruses, diagnostic applications and development of safer vaccine candidates.

Defective HIV-1 quasispecies in the form of multiply drug-resistant proviral DNA within cells can be rescued by superinfection with different subtype variants of HIV-1 and by HIV-2 and SIV.

OBJECTIVES: HIV-1 generates swarms of similar, but genetically distinct, variants termed quasispecies and many of these variants can be defective. A relevant question is whether such defective species can contribute to viral pathogenesis. Indeed, we previously reported that a presumed recombination of defective proviral DNA with other complementary defective proviral DNA or with wild-type viral DNA in the aftermath of superinfection could lead to the rescue of defective provirus and the production of replication-competent virus. We then wished to determine whether such rescue could be affected by viruses of different subtypes or even by other members of the retrovirus family. METHODS: Here, we have used drug resistance mutations within the HIV genome as markers of potential recombination. RESULTS: We show that a defective proviral DNA within cells can be rescued by the superinfection of MT2 cells by various subtypes of HIV-1, and by HIV-2 and simian immunodeficiency virus, but not by human T cell leukaemia virus type 1 or by human herpes virus-6. The drug-resistance phenotype of the rescued HIV was confirmed in a second round of infection. CONCLUSIONS: Defective proviral HIV-1 can be rescued by the infection by different variants of HIV-1 and by several other retroviruses as well.

Design requirements for interfering particles to maintain coadaptive stability with HIV-1.

Defective interfering particles (DIPs) are viral deletion mutants lacking essential transacting or packaging elements and must be complemented by wild-type virus to propagate. DIPs transmit through human populations, replicating at the expense of the wild-type virus and acting as molecular parasites of viruses. Consequently, engineered DIPs have been proposed as therapies for a number of diseases, including human immunodeficiency virus (HIV). However, it is not clear if DIP-based therapies would face evolutionary blocks given the high mutation rates and high within-host diversity of lentiviruses. Divergent evolution of HIV and DIPs appears likely since natural DIPs have not been detected for lentiviruses, despite extensive sequencing of HIVs and simian immunodeficiency viruses (SIVs). Here, we tested if the apparent lack of lentiviral DIPs is due to natural selection and analyzed which molecular characteristics a DIP or DIP-based therapy would need to maintain coadaptive stability with HIV-1. Using a well-established mathematical model of HIV-1 in a host extended to include its replication in a single cell and interference from DIP, we calculated evolutionary selection coefficients. The analysis predicts that interference by codimerization between DIPs and HIV-1 genomes is evolutionarily unstable, indicating that recombination between DIPs and HIV-1 would be selected against. In contrast, DIPs that interfere via competition for capsids have the potential to be evolutionarily stable if the capsid-to-genome production ratio of HIV-1 is >1. Thus, HIV-1 variants that attempt to "starve" DIPs to escape interference would be selected against. In summary, the analysis suggests specific experimental measurements that could address the apparent lack of naturally occurring lentiviral DIPs and specifies how therapeutic approaches based on engineered DIPs could be evolutionarily robust and avoid recombination.

Biological and genomic analysis of a PBSX-like defective phage induced from Bacillus pumilus AB94180.

Defective prophages, which are found in the genomes of many bacteria, are unable to complete a viral replication cycle and propagate in their hosts as healthy prophages. They package random DNA fragments derived from various sites of the host chromosome instead of their own genomes. In this study, we characterized a defective phage, PBP180, which was induced from Bacillus pumilus AB94180 by treatment with mitomycin C. Electron microscopy showed that the PBP180 particle has a head with a hexagonal outline of ~40 nm in diameter and a long tail. The DNA packaged in the PBP180 head consists of 8-kb DNA fragments from random portions of the host chromosome. The head and tail proteins of the PBP180 particle consist of four major proteins of approximately 49, 33, 16 and 14 kDa. The protein profile of PBP180 is different from that of PBSX, a well-known defective phage induced from Bacillus subtilis 168. A killing activity test against two susceptible strains each of B. subtilis and B. pumilus showed that the defective particles of PBP180 killed three strains other than its own host, B. pumilus AB94180, differing from the host-killing ranges of the defective phages PBSX, PBSZ (induced from B. subtilis W23), and PBSX4 (induced from B. pumilus AB94044). The genome of the PBP180 prophage, which is integrated in the B. pumilus AB94180 chromosome, is 28,205 bp in length, with 40 predicted open reading frames (ORFs). Further genomic comparison of prophages PBP180, PBSX, PBSZ and other PBSX-like prophage elements in B. pumilus strains revealed that their overall architectures are similar, but significant low homology exists in ORF29-ORF38, which presumably encode tail fiber proteins involved in recognition and killing of susceptible strains.

Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production.

During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-ß expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.

Differential activation of cellular DNA damage responses by replication-defective and replication-competent adenovirus mutants.

Adenovirus (Ad) mutants that lack early region 4 (E4) activate the phosphorylation of cellular DNA damage response proteins. In wild-type Ad type 5 (Ad5) infections, E1b and E4 proteins target the cellular DNA repair protein Mre11 for redistribution and degradation, thereby interfering with its ability to activate phosphorylation cascades important during DNA repair. The characteristics of Ad infection that activate cellular DNA repair processes are not yet well understood. We investigated the activation of DNA damage responses by a replication-defective Ad vector (AdRSVßgal) that lacks E1 and fails to produce the immediate-early E1a protein. E1a is important for activating early gene expression from the other viral early transcription units, including E4. AdRSVßgal can deliver its genome to the cell, but it is subsequently deficient for viral early gene expression and DNA replication. We studied the ability of AdRSVßgal-infected cells to induce cellular DNA damage responses. AdRSVßgal infection does activate formation of foci containing the Mdc1 protein. However, AdRSVßgal fails to activate phosphorylation of the damage response proteins Nbs1 and Chk1. We found that viral DNA replication is important for Nbs1 phosphorylation, suggesting that this step in the viral life cycle may provide an important trigger for activating at least some DNA repair proteins.

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.