Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4-9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV.

Production of high titer attenuated poliovirus strains on the serum-free PER.C6(®) cell culture platform for the generation of safe and affordable next generation IPV.

BACKGROUND: As poliovirus eradication draws closer, alternative Inactivated Poliovirus Vaccines (IPV) are needed to overcome the risks associated with continued use of the Oral Poliovirus Vaccine and of neurovirulent strains used during manufacture of conventional (c) IPV. We have previously demonstrated the susceptibility of the PER.C6(®) cell line to cIPV strains; here we investigated the suspension cell culture platform for growth of attenuated poliovirus strains. METHODS: We examined attenuated Sabin strain productivity on the PER.C6(®) cell platform compared to the conventional Vero cell platform. The suitability of the suspension cell platform for propagation of rationally-attenuated poliovirus strains (stabilized Sabin type 3 S19 derivatives and genetically attenuated and stabilized MonoCre(X) strains), was also assessed. Yields were quantified by infectious titer determination and D-antigen ELISA using either serotype-specific polyclonal rabbit sera for Sabin strains or monoclonal cIPV-strain-specific antibodies for cIPV, S19 and MonoCre(X) strains. RESULTS: PER.C6(®) cells supported the replication of Sabin strains to yields of infectious titers that were in the range of cIPV strains at 32.5°C. Sabin strains achieved 30-fold higher yields (p<0.0001) on the PER.C6(®) cell platform as compared to the Vero cell platform in infectious titer and D-antigen content. Furthermore, Sabin strain productivity on the PER.C6(®) cell platform was maintained at 10l scale. Yields of infectious titers of S19 and MonoCre(X) strains were 0.5-1 log10 lower than seen for cIPV strains, whereas D-antigen yield and productivities in doses/ml using rationally-attenuated strains were in line with yields reported for cIPV strains. CONCLUSIONS: Sabin and rationally-attenuated polioviruses can be grown to high infectious titers and D-antigen yields. Sabin strain infection shows increased productivity on the PER.C6(®) cell platform as compared to the conventional Vero cell platform. Novel cell platforms with the potential for higher yields could contribute to increased affordability of a next generation of IPV vaccines needed for achieving and maintaining poliovirus eradication.

Synthetic virus seeds for improved vaccine safety: Genetic reconstruction of poliovirus seeds for a PER.C6 cell based inactivated poliovirus vaccine.

Safety of vaccines can be compromised by contamination with adventitious agents. One potential source of adventitious agents is a vaccine seed, typically derived from historic clinical isolates with poorly defined origins. Here we generated synthetic poliovirus seeds derived from chemically synthesized DNA plasmids encoding the sequence of wild-type poliovirus strains used in marketed inactivated poliovirus vaccines. The synthetic strains were phenotypically identical to wild-type polioviruses as shown by equivalent infectious titers in culture supernatant and antigenic content, even when infection cultures are scaled up to 10-25L bioreactors. Moreover, the synthetic seeds were genetically stable upon extended passaging on the PER.C6 cell culture platform. Use of synthetic seeds produced on the serum-free PER.C6 cell platform ensures a perfectly documented seed history and maximum control over starting materials. It provides an opportunity to maximize vaccine safety which increases the prospect of a vaccine end product that is free from adventitious agents.

Brunenders: a partially attenuated historic poliovirus type I vaccine strain.

Brunenders, a type I poliovirus (PV) strain, was developed in 1952 by J. F. Enders and colleagues through serial in vitro passaging of the parental Brunhilde strain, and was reported to display partial neuroattenuation in monkeys. This phenotype of attenuation encouraged two vaccine manufacturers to adopt Brunenders as the type I component for their inactivated poliovirus vaccines (IPVs) in the 1950s, although today no licensed IPV vaccine contains Brunenders. Here we confirmed, in a transgenic mouse model, the report of Enders on the reduced neurovirulence of Brunenders. Although dramatically neuroattenuated relative to WT PV strains, Brunenders remains more virulent than the attenuated oral vaccine strain, Sabin 1. Importantly, the neuroattenuation of Brunenders does not affect in vitro growth kinetics and in vitro antigenicity, which were similar to those of Mahoney, the conventional type I IPV vaccine strain. We showed, by full nucleotide sequencing, that Brunhilde and Brunenders differ at 31 nucleotides, eight of which lead to amino acid changes, all located in the capsid. Upon exchanging the Brunenders capsid sequence with that of the Mahoney capsid, WT neurovirulence was regained in vivo, suggesting a role for the capsid mutations in Brunenders attenuation. To date, as polio eradication draws closer, the switch to using attenuated strains for IPV is actively being pursued. Brunenders preceded this novel strategy as a partially attenuated IPV strain, accompanied by decades of successful use in the field. Providing data on the attenuation of Brunenders may be of value in the further construction of attenuated PV strains to support the grand pursuit of the global eradication of poliomyelitis.

PER.C6(®) cells as a serum-free suspension cell platform for the production of high titer poliovirus: a potential low cost of goods option for world supply of inactivated poliovirus vaccine.

There are two highly efficacious poliovirus vaccines: Sabin's live-attenuated oral polio vaccine (OPV) and Salk's inactivated polio vaccine (IPV). OPV can be made at low costs per dose and is easily administrated. However, the major drawback is the frequent reversion of the OPV vaccine strains to virulent poliovirus strains which can result in Vaccine Associated Paralytic Poliomyelitis (VAPP) in vaccinees. Furthermore, some OPV revertants with high transmissibility can circulate in the population as circulating Vaccine Derived Polioviruses (cVDPVs). IPV does not convey VAPP and cVDPVs but the high costs per dose and insufficient supply have rendered IPV an unfavorable option for low and middle-income countries. Here, we explored whether the human PER.C6(®) cell-line, which has the unique capability to grow at high density in suspension, under serum-free conditions, could be used as a platform for high yield production of poliovirus. PER.C6(®) cells supported replication of all three poliovirus serotypes with virus titers ranging from 9.4 log(10) to 11.1 log(10)TCID(50)/ml irrespective of the volume scale (10 ml in shaker flasks to 2 L in bioreactors). This production yield was 10-30 fold higher than in Vero cell cultures performed here, and even 100-fold higher than what has been reported for Vero cell cultures in literature [38]. In agreement, the D-antigen content per volume PER.C6(®)-derived poliovirus was on average 30-fold higher than Vero-derived poliovirus. Interestingly, PER.C6(®) cells produced on average 2.5-fold more D-antigen units per cell than Vero cells. Based on our findings, we are exploring PER.C6(®) as an interesting platform for large-scale production of poliovirus at low costs, potentially providing the basis for global supply of an affordable IPV.

Reconstructing the dynamics of HIV evolution within hosts from serial deep sequence data.

At the early stage of infection, human immunodeficiency virus (HIV)-1 predominantly uses the CCR5 coreceptor for host cell entry. The subsequent emergence of HIV variants that use the CXCR4 coreceptor in roughly half of all infections is associated with an accelerated decline of CD4+ T-cells and rate of progression to AIDS. The presence of a 'fitness valley' separating CCR5- and CXCR4-using genotypes is postulated to be a biological determinant of whether the HIV coreceptor switch occurs. Using phylogenetic methods to reconstruct the evolutionary dynamics of HIV within hosts enables us to discriminate between competing models of this process. We have developed a phylogenetic pipeline for the molecular clock analysis, ancestral reconstruction, and visualization of deep sequence data. These data were generated by next-generation sequencing of HIV RNA extracted from longitudinal serum samples (median 7 time points) from 8 untreated subjects with chronic HIV infections (Amsterdam Cohort Studies on HIV-1 infection and AIDS). We used the known dates of sampling to directly estimate rates of evolution and to map ancestral mutations to a reconstructed timeline in units of days. HIV coreceptor usage was predicted from reconstructed ancestral sequences using the geno2pheno algorithm. We determined that the first mutations contributing to CXCR4 use emerged about 16 (per subject range 4 to 30) months before the earliest predicted CXCR4-using ancestor, which preceded the first positive cell-based assay of CXCR4 usage by 10 (range 5 to 25) months. CXCR4 usage arose in multiple lineages within 5 of 8 subjects, and ancestral lineages following alternate mutational pathways before going extinct were common. We observed highly patient-specific distributions and time-scales of mutation accumulation, implying that the role of a fitness valley is contingent on the genotype of the transmitted variant.

Longitudinal analysis of early HIV-1-specific neutralizing activity in an elite neutralizer and in five patients who developed cross-reactive neutralizing activity.

We previously established that at 3 years postseroconversion, ~30% of HIV-infected individuals have cross-reactive neutralizing activity (CrNA) in their sera. Here we studied the kinetics with which CrNA develops and how these relate to the development of autologous neutralizing activity as well as viral escape and diversification. For this purpose, sera from five individuals with CrNA and one elite neutralizer that were obtained at three monthly intervals in the first year after seroconversion and at multiple intervals over the disease course were tested for neutralizing activity against an established multiclade panel of six viruses. The same serum samples, as well as sera from three individuals who lacked CrNA, were tested for their neutralizing activities against autologous clonal HIV-1 variants from multiple time points covering the disease course from seroconversion onward. The elite neutralizer already had CrNA at 9.8 months postseroconversion, in contrast with the findings for the other five patients, in whom CrNA was first detected at 20 to 35 months postseroconversion and peaked around 35 months postseroconversion. In all patients, CrNA coincided with neutralizing activity against autologous viruses that were isolated <12 months postseroconversion, while viruses from later time points had already escaped autologous neutralizing activity. Also, the peak in gp160 sequence diversity coincided with the peak of CrNA titers. Individuals who lacked CrNA had lower peak autologous neutralizing titers, viral escape, and sequence diversity than individuals with CrNA. A better understanding of the underlying factors that determine the presence of CrNA or even an elite neutralizer phenotype may aid in the design of an HIV-1 vaccine.

The evolution of human immunodeficiency virus type-1 (HIV-1) envelope molecular properties and coreceptor use at all stages of infection in an HIV-1 donor-recipient pair.

To trace the evolutionary patterns underlying evolution of coreceptor use within a host, we studied an HIV-1 transmission pair involving a donor who exclusively harbored CCR5-using (R5) variants throughout his entire disease course and a recipient who developed CXCR4-using variants. Over time, R5 variants in the donor optimized coreceptor use, which was associated with an increased number of potential N-linked glycosylation sites (PNGS) and elevated V3 charge in the viral envelope. Interestingly, R5 variants that were transmitted to the recipient preserved the viral characteristics of this late stage genotype and phenotype. Following a selective sweep, CXCR4-using variants subsequently emerged in the recipient coinciding with a further increase in the number of PNGS and V3 charge in the envelope of R5 viruses. Although described in a single transmission pair, the transmission and subsequent persistence of R5 variants with late stage characteristics demonstrate the potential for coreceptor use adaptation at the population level.

Detection of inferred CCR5- and CXCR4-using HIV-1 variants and evolutionary intermediates using ultra-deep pyrosequencing.

The emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants is associated with accelerated disease progression. CXCR4-using variants are believed to evolve from CCR5-using variants, but due to the extremely low frequency at which transitional intermediate variants are often present, the kinetics and mutational pathways involved in this process have been difficult to study and are therefore poorly understood. Here, we used ultra-deep sequencing of the V3 loop of the viral envelope in combination with the V3-based coreceptor prediction tools PSSM(NSI/SI) and geno2pheno([coreceptor]) to detect HIV-1 variants during the transition from CCR5- to CXCR4-usage. We analyzed PBMC and serum samples obtained from eight HIV-1-infected individuals at three-month intervals up to one year prior to the first phenotypic detection of CXCR4-using variants in the MT-2 assay. Between 3,482 and 10,521 reads were generated from each sample. In all individuals, V3 sequences of predicted CXCR4-using HIV-1 were detected at least three months prior to phenotypic detection of CXCR4-using variants in the MT-2 assay. Subsequent analysis of the genetic relationships of these V3 sequences using minimum spanning trees revealed that the transition in coreceptor usage followed a stepwise mutational pathway involving sequential intermediate variants, which were generally present at relatively low frequencies compared to the major predicted CCR5- and CXCR4-using variants. In addition, we observed differences between individuals with respect to the number of predicted CXCR4-using variants, the diversity among major predicted CCR5-using variants, and the presence or absence of intermediate variants with discordant phenotype predictions. These results provide the first detailed description of the mutational pathways in V3 during the transition from CCR5- to CXCR4-usage in natural HIV-1 infection.

Evolution of human immunodeficiency virus type 1 in a patient with cross-reactive neutralizing activity in serum.

Analysis of longitudinally obtained HIV-1 env sequences from an individual with reported cross-reactive neutralizing activity revealed that the majority of viral variants obtained from serum between 4 and 7 years after seroconversion were unable to persist in peripheral blood. Here we show that these viral variants were more sensitive to autologous serum neutralization, had shorter envelopes with fewer potential N-linked glycosylation sites, and showed lower replication kinetics than successfully evolving HIV-1 variants. These data reflect the host selection pressures on phenotypic characteristics of HIV-1 and illustrate in detail the dynamic interaction between HIV-1 and its host's humoral immune responses.

Comparison of in vivo and in vitro evolution of CCR5 to CXCR4 coreceptor use of primary human immunodeficiency virus type 1 variants.

During the course of at least 50% of HIV-1 subtype B infections, CCR5-using (R5) viruses evolve towards a CXCR4-using phenotype. To gain insight in the transition from CCR5 to CXCR4 coreceptor use, we investigated whether acquisition of CXCR4 use in vitro of R5 viruses from four patients resembled this process in vivo. R5 variants from only one patient acquired CXCR4 use in vitro. These variants had envelopes with higher V3 charge and higher number of potential N-linked glycosylation sites when compared to R5 variants that failed to gain CXCR4 use in vitro. In this patient, acquisition of CXCR4 use in vitro and in vivo was associated with multiple mutational patterns not necessarily involving the V3 region. However, changes at specific V3 positions were prerequisite for persistence of CXCR4-using variants in vivo, suggesting that positive selection targeting the V3 loop is required for emergence of CXCR4-using variants during natural disease course.

Impact of CCR5delta32 host genetic background and disease progression on HIV-1 intrahost evolutionary processes: efficient hypothesis testing through hierarchical phylogenetic models.

The interplay between C-C chemokine receptor type 5 (CCR5) host genetic background, disease progression, and intrahost HIV-1 evolutionary dynamics remains unclear because differences in viral evolution between hosts limit the ability to draw conclusions across hosts stratified into clinically relevant populations. Similar inference problems are proliferating across many measurably evolving pathogens for which intrahost sequence samples are readily available. To this end, we propose novel hierarchical phylogenetic models (HPMs) that incorporate fixed effects to test for differences in dynamics across host populations in a formal statistical framework employing stochastic search variable selection and model averaging. To clarify the role of CCR5 host genetic background and disease progression on viral evolutionary patterns, we obtain gp120 envelope sequences from clonal HIV-1 variants isolated at multiple time points in the course of infection from populations of HIV-1-infected individuals who only harbored CCR5-using HIV-1 variants at all time points. Presence or absence of a CCR5 wt/Δ32 genotype and progressive or long-term nonprogressive course of infection stratify the clinical populations in a two-way design. As compared with the standard approach of analyzing sequences from each patient independently, the HPM provides more efficient estimation of evolutionary parameters such as nucleotide substitution rates and d(N)/d(S) rate ratios, as shown by significant shrinkage of the estimator variance. The fixed effects also correct for nonindependence of data between populations and results in even further shrinkage of individual patient estimates. Model selection suggests an association between nucleotide substitution rate and disease progression, but a role for CCR5 genotype remains elusive. Given the absence of clear d(N)/d(S) differences between patient groups, delayed onset of AIDS symptoms appears to be solely associated with lower viral replication rates rather than with differences in selection on amino acid fixation.

Genetic composition of replication competent clonal HIV-1 variants isolated from peripheral blood mononuclear cells (PBMC), HIV-1 proviral DNA from PBMC and HIV-1 RNA in serum in the course of HIV-1 infection.

The HIV-1 quasispecies in peripheral blood mononuclear cells (PBMC) is considered to be a mix of actively replicating, latent, and archived viruses and may be genetically distinct from HIV-1 variants in plasma that are considered to be recently produced. Here we analyzed the genetic relationship between gp160 env sequences from replication competent clonal HIV-1 variants that were isolated from PBMC and from contemporaneous HIV-1 RNA in serum and HIV-1 proviral DNA in PBMC of four longitudinally studied therapy naïve HIV-1 infected individuals. Replication competent clonal HIV-1 variants, HIV-1 RNA from serum, and HIV-1 proviral DNA from PBMC formed a single virus population at most time points analyzed. However, an under-representation in serum of HIV-1 sequences with predicted CXCR4 usage was sometimes observed implying that the analysis of viral sequences from different sources may provide a more complete assessment of the viral quasispecies in peripheral blood in vivo.

High prevalence of neutralizing activity against multiple unrelated human immunodeficiency virus type 1 (HIV-1) subtype B variants in sera from HIV-1 subtype B-infected individuals: evidence for subtype-specific rather than strain-specific neutralizing activity.

It is assumed that an effective human immunodeficiency virus type 1 (HIV-1) vaccine should be capable of eliciting neutralizing antibodies. However, even the best antibodies known to date lack neutralizing ability against a significant proportion of primary HIV-1 variants and, despite great efforts, still no immunogen is available that can elicit humoral immunity which is protective against infection or disease progression. We tested sera from 35 participants in the Amsterdam Cohort Studies on HIV-1 infection, who were all infected with HIV-1 subtype B and therapy-naïve at the time of sampling, for neutralizing activity against a panel of 23 tier 2-3 HIV-1 variants, with a minimum of five HIV-1 variants per subtype (A, B, C and D). Strong cross-clade neutralizing activity was detected in sera from seven individuals. Strikingly, sera from 22 of 35 individuals (63%) neutralized three or more of the six tier 2-3 HIV-1 subtype B viruses in the panel. There was a strong correlation between neutralization titre and breadth in serum. Indeed, the IC(50) of sera with strong cross-clade neutralizing activity was significantly higher than the IC(50) of sera with cross-subtype B activity, which, in turn, had a higher IC(50) than sera with the lowest neutralization breadth. These results imply that humoral immunity, at least in HIV-1 subtype B-infected individuals, is often subtype-specific rather than strain-specific and that the breadth of neutralization is correlated with the titre of neutralizing activity in serum. Considering the difficulties in designing a vaccine that is capable of eliciting cross-clade neutralizing activity, subtype-specific vaccines may be explored as an interesting alternative.