Emergence Of Escape Mutants Research Articles

Characterisation of the antibody-mediated selective pressure driving intra-host evolution of SARS-CoV-2 in prolonged infection.

Neutralising antibodies against the SARS-CoV-2 spike (S) protein are major determinants of protective immunity, though insufficient antibody responses may cause the emergence of escape mutants. We studied the humoral immune response causing intra-host evolution in a B-cell depleted, haemato-oncologic patient experiencing clinically severe, prolonged SARS-CoV-2 infection with a virus of lineage B.1.177.81. Following bamlanivimab treatment at an early stage of infection, the patient developed a bamlanivimab-resistant mutation, S:S494P. After five weeks of apparent genetic stability, the emergence of additional substitutions and deletions within the N-terminal domain (NTD) and the receptor binding domain (RBD) of S was observed. Notably, the composition and frequency of escape mutations changed in a short period with an unprecedented dynamic. The triple mutant S:Delta141-4 E484K S494P became dominant until virus elimination. Routine serology revealed no evidence of an antibody response in the patient. A detailed analysis of the variant-specific immune response by pseudotyped virus neutralisation test, surrogate virus neutralisation test, and immunoglobulin-capture enzyme immunoassay showed that the onset of an IgM-dominated antibody response coincided with the appearance of escape mutations. The formation of neutralising antibodies against S:Delta141-4 E484K S494P correlated with virus elimination. One year later, the patient experienced clinically mild re-infection with Omicron BA.1.18, which was treated with sotrovimab and resulted in an increase in Omicron-reactive antibodies. In conclusion, the onset of an IgM-dominated endogenous immune response in an immunocompromised patient coincided with the appearance of additional mutations in the NTD and RBD of S in a bamlanivimab-resistant virus. Although virus elimination was ultimately achieved, this humoral immune response escaped detection by routine diagnosis and created a situation temporarily favouring the rapid emergence of various antibody escape mutants with known epidemiological relevance.

Genomic Evolution and Surveillance of Respiratory Syncytial Virus during the 2023-2024 Season.

Respiratory syncytial virus (RSV) is a significant cause of morbidity, particularly in infants. This study describes RSV genomic diversity and disease outcomes during the 2023-2024 season in the Johns Hopkins Hospital System (JHHS). Between August and December 2023, 406 patient samples were sequenced, showing that RSV-B GB5.0.5a was the dominant genotype detected. RSV-A genotype GA2.3.5 was detected less frequently. Metadata analysis of patient data revealed that, although RSV-B was more commonly detected, patients with RSV-A infections were more frequently hospitalized. Analysis of both the G- and F-genes revealed multiple amino acid substitutions in both RSV-A and RSV-B, with some positions within the F-protein that could be associated with evasion of antibody responses. Phylogenetic analysis revealed the genetic diversity of circulating GB5.0.5a and GA2.3.5 genotypes. This study serves as an important baseline for genomic surveillance of RSV within the JHHS and will assist in characterizing the impact of the newly approved RSV vaccines on RSV genomic evolution and the emergence of escape mutations.

Abstract 2730: Beyond antibodies and CAR-T: Topologically-engineered, super-dimeric antibody-like molecules with dual Fc domains for trispecific, bivalent targeting of CD19, CD20, and Fcgamma receptors

Abstract Background: Depletion of B cells has resulted in major therapeutic benefits in autoimmune diseases and hematological malignancies. Several FDA-approved B cell-depleting antibody-based therapies are available, including antibody-drug conjugates, T cell engagers, CAR-T cells, and antibody variants with enhanced effector functions through Fc engineering. However, a significant unmet need remains in both oncology and autoimmune disease. All currently approved B cell-depleting therapies target a single B cell antigen, increasing potential for incomplete depletion and emergence of escape mutants that give rise to relapse and treatment failure. Methods: Here we describe trispecific antibody-like molecules generated using GEM-DIMER technology. In addition to bivalent target binding of both CD19 and CD20, the GEM-DIMER candidates are designed to have two Fc domains to enable powerful effector functions via cooperative binding of Fcgamma receptors. The approach is based on incorporation of a super-dimerization domain into the hinge region of an immunoglobulin heavy chain, enabling the combination of all of the components of two IgG antibodies into a single molecule. Using this approach, we generated GEM-DIMER candidates from rituximab and FMC63, the parental anti-CD19 antibody for anti-CD19 scFv used in approved CAR-T cell therapies. Results: CD19/CD20-targeting GEM-DIMER molecules demonstrated binding to both CD19 and CD20 with affinities comparable to the individual parent antibodies. Importantly, CD19/CD20-targeting GEM-DIMER molecules demonstrated robust depletion of human B cells in overnight cultures of whole blood. Moreover, antibody-dependent cellular cytotoxicity (ADCC) was demonstrated in co-cultures of the human B cell lymphoma cell line Raji and human peripheral blood mononuclear cells (PBMC). As expected, due to the presence of dual Fc domains, CD19/CD20-targeting GEM-DIMER molecules exhibited enhanced binding to Fcgamma receptors, further explaining the robust effector functions observed. Conclusions: CD19/CD20-targeting GEM-DIMER molecules are promising candidates to provide efficient depletion of both CD19+ and CD20+ cells, providing potential for broad and deep depletion of B cells with reduced risk of emergence of antigen escape variants. These data support the advancement of CD19/CD20-targeting GEM-DIMER molecules in multiple indications where depletion of CD19+ and/or CD20+ B cells is needed. Preparations for clinical investigation are ongoing. Citation Format: Daniel J. Capon, Nelson L. Chan, Larisa Troitskaya, Marina Fomin, Ursula Edman, Brendon Frank, Benjamin Z. Capon, Brian Law, Steven J. Chapin, Gavin M. Lewis, Malcolm L. Gefter, Juha Punnonen. Beyond antibodies and CAR-T: Topologically-engineered, super-dimeric antibody-like molecules with dual Fc domains for trispecific, bivalent targeting of CD19, CD20, and Fcgamma receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2730.

Emergence and dissemination of equine-like G3P[8] rotavirus A in Brazil between 2015 and 2021.

Rotavirus A (RVA) is a major cause of acute gastroenteritis globally that is classically genotyped by its two immunodominant outer capsid proteins, VP7 (G-) and VP4 (P-). Recent evidence suggests that the reassortant equine-like G3P[8] strain played a substantial role in RVA transmission in Brazil since 2015. To understand its global emergence and dissemination in Brazilian territory, stool samples collected from 11 Brazilian states (n = 919) were genotyped by RT-qPCR and proceeded to sequence the VP7 gene (n = 102, 79 being newly generated) of the G3P[8] samples with pronounced viral loads. Our phylogenetic genotyping showed that G3P[8] became the dominant strain in Brazil between 2017 and 2020, with equine-like variants representing 75%-100% of VP7 samples in this period. A Bayesian discrete phylogeographic analysis strongly suggests that the equine-like G3P[8] strain originated in Asia during the early 2010s and subsequently spread to Europe, the Caribbean, and South America. Multiple introductions were detected in Brazil between 2014 and 2017, resulting in five national clusters. The reconstruction of the effective population size of the largest Brazilian cluster showed an expansion until 2017, followed by a plateau phase until 2019 and subsequent contraction. Our study also supports that most mutations fixed during equine-like G3P[8] evolution were synonymous, suggesting that adaptive evolution was not an important driving force during viral dissemination in humans, potentially increasing its susceptibility to acquired immunity. This research emphasizes the need for comprehensive rotavirus genomic surveillance that allows close monitoring of its ever-shifting composition and informs more effective public health policies.IMPORTANCEOur original article demonstrated the origin and spread in a short time of equine-like G3P[8] in Brazil and the world. Due to its segmented genome, it allows numerous mechanisms including genetic drift and reassortment contribute substantially to the genetic diversity of rotavirus. Although the effectiveness and increasing implementation of vaccination have not been questioned, a matter of concern is its impact on the emergence of escape mutants or even the spread of unusual strains of zoonotic transmission that could drive epidemic patterns worldwide. This research emphasizes the need for comprehensive rotavirus genomic surveillance, which could facilitate the formulation of public policies aimed at preventing and mitigating its transmission.

Rapid Selection of Sotrovimab Escape Variants in Severe Acute Respiratory Syndrome Coronavirus 2 Omicron-Infected Immunocompromised Patients.

Monoclonal antibodies (mAbs) that target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are predominantly less effective against Omicron variants. Immunocompromised patients often experience prolonged viral shedding, resulting in an increased risk of viral escape. In an observational, prospective cohort, 57 patients infected with Omicron variants who received sotrovimab alone or in combination with remdesivir were followed. The study end points were a decrease in SARS-CoV-2 RNA <106 copies/mL in nasopharyngeal swabs at day 21 and the emergence of escape mutations at days 7, 14, and 21 after sotrovimab administration. All SARS-CoV-2 samples were analyzed using whole-genome sequencing. Individual variants within the quasispecies were subsequently quantified and further characterized using a pseudovirus neutralization assay. The majority of patients (43 of 57, 75.4%) were immunodeficient, predominantly due to immunosuppression after organ transplantation or hematologic malignancies. Infections by Omicron/BA.1 comprised 82.5%, while 17.5% were infected by Omicron/BA.2. Twenty-one days after sotrovimab administration, 12 of 43 (27.9%) immunodeficient patients had prolonged viral shedding compared with 1 of 14 (7.1%) immunocompetent patients (P = .011). Viral spike protein mutations, some specific for Omicron (e.g., P337S and/or E340D/V), emerged in 14 of 43 (32.6%) immunodeficient patients, substantially reducing sensitivity to sotrovimab in a pseudovirus neutralization assay. Combination therapy with remdesivir significantly reduced emergence of escape variants. Immunocompromised patients face a considerable risk of prolonged viral shedding and emergence of escape mutations after early therapy with sotrovimab. These findings underscore the importance of careful monitoring and the need for dedicated clinical trials in this patient population.

Abrogation of Marek\u2019s disease virus replication using CRISPR/Cas9

Marek’s disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.

A potent human neutralizing antibody Fc-dependently reduces established HBV infections.

Hepatitis B virus (HBV) infection is a major global health problem. Currently-available therapies are ineffective in curing chronic HBV infection. HBV and its satellite hepatitis D virus (HDV) infect hepatocytes via binding of the preS1 domain of its large envelope protein to sodium taurocholate cotransporting polypeptide (NTCP). Here, we developed novel human monoclonal antibodies that block the engagement of preS1 with NTCP and neutralize HBV and HDV with high potency. One antibody, 2H5-A14, functions at picomolar level and exhibited neutralization-activity-mediated prophylactic effects. It also acts therapeutically by eliciting antibody-Fc-dependent immunological effector functions that impose durable suppression of viral infection in HBV-infected mice, resulting in reductions in the levels of the small envelope antigen and viral DNA, with no emergence of escape mutants. Our results illustrate a novel antibody-Fc-dependent approach for HBV treatment and suggest 2H5-A14 as a novel clinical candidate for HBV prevention and treatment of chronic HBV infection.

A comparative characteristic of antigenic properties of recombinant and native hbs-antigens with G145R mutation and evaluation of their immunogenicity

One of the important reasons for spreading of hepatitis B virus (HBV) under conditions of vaccinepressure is emergence of escape mutations. Prevalent G145R mutation in S-gene leads to the most expressed changes of serological properties of HBV. Consequently, HBsAg is modifed so thoroughly that it cannot be recognized by the majority of anti-HBs. Mutant G145R also differs from a wild type HBsAg by its immunogenic properties. At present, the relevance of enhancement of hepatitis B vaccine in view of mutant virus variants has been recognized. a comparative study of antigenic and immunogenic properties of native and recombinant G145R mutants and an estimation of possibility for developing antigenic component of hepatitis B vaccine with G145R mutation in HBsAg. antigenic properties of recombinant HBsAg with G145R mutation were compared with each other and with native mutants by serological fngerprinting method. Then, BALB/c mice and sheep were immunized with selected recombinant antigen under different protocols. Titers of antibodies specifc to wild type or mutant G145R type of HBsAg in sera of immunized animals were measured. it was found that not all the recombinant HBsAg variants with G145R substitution have the same antigenic properties as native HBsAg with similar mutation. Recombinant HBsAg selected according to the principle of antigenic similarity possesses immunogenicity in mice and sheep causing the production of antibodies reacting with native wild and mutant type HBsAg. It was shown that mutant antigen is less immunogenic, requires larger doses and more time for the development of immune response; however, it is capable of causing an antibody level comparable with wild type antigen. preliminary selection of recombinant HBsAg containing G145R mutation with antigenic and immunogenic properties similar to the native analogue creates the basis for development of a specifc component of hepatitis B vaccine with escape mutation G145R in HBsAg.

Occult hepatitis B virus infection and S gene escape mutants in HIV-infected patients after hepatitis B virus vaccination.

Hepatitis B virus (HBV) vaccination is recommended for HIV patients. Despite the relative success of HBV vaccination, breakthrough infections can occur infrequently in patients, and it can be due to occult HBV infection, vaccine unresponsiveness and/or emergence of escape mutants. This study assessed the presence of occult HBV infection and S gene escape mutants in HIV-positive patients after HBV vaccination. Ninety-two HIV-positive patients were enrolled in this study, including 52 responders to HBV vaccine and 40 non-responders. All of the cases received HBV vaccine according to routine HBV vaccination protocols. The presence of HBV-DNA was determined by real-time polymerase chain reaction (PCR). In HBV-DNA positive samples, the most conserved regions of S gene sequences were amplified by nested PCR and PCR products were sequenced. Occult HBV infection was detected in two cases. Glycine to arginine mutation at residue 145 (G145R) within the 'a' region of the S gene was detected in one of the occult HBV infection cases who was in the non-responder group. This study showed that the prevalence of occult HBV infection and vaccine escape mutants was low in our HBV-vaccinated HIV-positive patients in both responder and non-responder groups, so there was no alarming evidence indicating breakthrough HBV infection in our vaccinated HIV-positive cases.

Molecular basis for universal HLA-A*0201–restricted CD8+ T-cell immunity against influenza viruses

Memory CD8(+)T lymphocytes (CTLs) specific for antigenic peptides derived from internal viral proteins confer broad protection against distinct strains of influenza A virus (IAV). However, immune efficacy can be undermined by the emergence of escape mutants. To determine how T-cell receptor (TCR) composition relates to IAV epitope variability, we used ex vivo peptide-HLA tetramer enrichment and single-cell multiplex analysis to compare TCRs targeted to the largely conserved HLA-A*0201-M158and the hypervariable HLA-B*3501-NP418antigens. The TCRαβs for HLA-B*3501-NP418 (+)CTLs varied among individuals and across IAV strains, indicating that a range of mutated peptides will prime different NP418-specific CTL sets. Conversely, a dominant public TRAV27/TRBV19(+)TCRαβ was selected in HLA-A*0201(+)donors responding to M158 This public TCR cross-recognized naturally occurring M158variants complexed with HLA-A*0201. Ternary structures showed that induced-fit molecular mimicry underpins TRAV27/TRBV19(+)TCR specificity for the WT and mutant M158peptides, suggesting the possibility of universal CTL immunity in HLA-A*0201(+)individuals. Combined with the high population frequency of HLA-A*0201, these data potentially explain the relative conservation of M158 Moreover, our results suggest that vaccination strategies aimed at generating broad protection should incorporate variant peptides to elicit cross-reactive responses against other specificities, especially those that may be relatively infrequent among IAV-primed memory CTLs.

Vaccine-Induced Plasma IgA Specific for the C1 Region of the HIV-1 Envelope Blocks Binding and Effector Function of IgG

A recently completed Phase III vaccine trial demonstrated a 32% estimated efficacy. An earlier analysis suggested that envelope (Env)-specific antibody responses were associated with a lower risk of infection. Of particular interest, anti-HIV Env immunoglobulin A (IgA) antibodies correlated with increased infection risk. The purpose of this study was to evaluate potential mechanisms of this phenomenon.Phase III controlled evaluation of the RV144 HIV vaccine to prevent HIV infection was conducted in Thailand. The current study represents a substudy in which all participants provided written consent to have plasma and cellular samples stored and subsequently tested.The levels and affinities of IgA and immunoglobulin G (IgG) antibodies for HIV Env proteins were measured. The ability of patient IgA as well as patient-derived IgA monoclonal antibodies to inhibit natural killer cell–mediated, antibody-dependent cell-mediated cytotoxicity (ADCC) was measured.The Env-specific IgA/IgG ratio directly correlated with infection risk, suggesting that the presence of IgA antibody directly inhibited ADCC function. Furthermore, IgA monoclonal antibodies derived from vaccines inhibited binding and blocked ADCC function. The phenomenon of interference of IgG function by IgA antibodies has been reported previously in other settings.Postvaccination polyclonal antibody responses vary among individuals. Those generating an excess of Env-specific IgA seem to be at increased risk for infection secondary to reduced ADCC effector function of natural killer cells.HIV has evolved multiple mechanisms to evade immune detection and elimination. These include impairment of neutralizing antibody generation, emergence of escape mutants, and Env glycosylation and conformational shielding. The vaccine studied in this article resulted in “blocking IgA antibodies” in a number of recipients and thus adds another level of complexity to vaccine design.

Hepatitis B virus surface antigen (HBsAg) mutations are rare but clustered in immune epitopes in chronic carriers from Sistan-Balouchestan Province, Iran.

Hepatitis B virus (HBV) gene and protein variations have frequently been observed in chronic patients. The aims of this study were to determine the genotypes as well as the patterns of HBsAg variations in chronically-infected patients from the south-eastern part of Iran. Twenty- one chronic inactive HBV carriers from Sistan-Balouchestan Province (an area with a low prevalence of HBV complications such as cirrhosis and hepatocellular carcinoma [HCC]) were enrolled. The surface genes were amplified, sequenced, and subsequently aligned using international and national Iranian database. All strains belonged to genotype D, subgenotype D1, and subtype ayw2. Of all 39 mutations occurred at 31 nucleotide positions, 15 (38.5%) were missense (amino acid altering) and 24 (61.5%) were silent (no amino acid changing). At the amino acid level, 15 substitutions occurred; 10 (66.67%) were distributed in different immune epitopes, five of which (33.33%) were in B cell epitopes; four (36.27%) were distributed in T helper epitopes, and one (6.67%) occurred inside CTL epitopes. A narrowly-focused immune pressure has been on the surface proteins, especially at the B cell level, led to the emergence of escape mutants in these patients that might be related to the pathogenicity of HBV chronic infection. Also, due to the negative selection imposed on HBV genome and the uniqueness of genotype D in this ethnic group, complications (cirrhosis and HCC) are lower than other published studies.

Splenic natural killer cells act early to select viral escape mutants during murine cytomegalovirus infection (P6135)

Abstract The large double-stranded DNA genome of murine cytomegalovirus (MCMV) is stable both in vitro and in vivo in the absence of immune pressure. However, one month after inoculation of plaque-purified MCMV into T cell-deficient mice, viral clones emerge with mutations in the viral MHC class I-like molecule, m157, due to selection by natural killer (NK) cells. While the emergence of escape mutants is dependent on the cognate interaction of m157 with the Ly49H NK cell activation receptor, the mechanism for mutant virus selection has not been fully dissected. Surprisingly, we show here that diverse mutations were already present in the salivary glands of C57BL/6 mice 14 days post-infection, much earlier than previously reported. Upon re-inoculation, MCMV was then subjected to purifying selection with one dominant clone emerging. To address the role of NK cells and the specific organs involved, we designed a single nucleotide polymorphism genotyping assay to quantify selection for a mutant virus with a single point mutation in m157 during a mixed infection. Remarkably, our results suggest that the escape mutant was initially selected in the spleen, but not the liver, as early as 2 days post-infection. Together, these findings suggest that the selective pressure exerted by NK cells on MCMV occurs primarily in the spleen during the initial rounds of viral replication, indicating that the host innate immune system exerts pressure on the virus almost immediately upon infection.

Genotypic but not phenotypic historical contingency revealed by viral experimental evolution

BackgroundThe importance of historical contingency in determining the potential of viral populations to evolve has been largely unappreciated. Identifying the constraints imposed by past adaptations is, however, of importance for understanding many questions in evolutionary biology, such as the evolution of host usage dynamics by multi-host viruses or the emergence of escape mutants that persist in the absence of antiviral treatments. To address this issue, we undertook an experimental approach in which sixty lineages of Tobacco etch potyvirus that differ in their past evolutionary history and degree of adaptation to Nicotiana tabacum were allowed to adapt to this host for 15 rounds of within host multiplication and transfer. We thereafter evaluated the degree of adaptation to the new host as well as to the original ones and characterized the consensus sequence of each lineage.ResultsWe found that past evolutionary history did not determine the phenotypic outcome of this common host evolution phase, and that the signal of local adaptation to past hosts had largely disappeared. By contrast, evolutionary history left footprints at the genotypic level, since the majority of host-specific mutations present at the beginning of this experiment were retained in the end-point populations and may have affected which new mutations were consequently fixed. This resulted in further divergence between the sequences despite a shared selective environment.ConclusionsThe present experiment reinforces the idea that the answer to the question “How important is historical contingency in evolution?” strongly depends on the level of integration of the traits studied. A strong historical contingency was found for TEV genotype, whereas a weak effect of on phenotypic evolution was revealed. In an applied context, our results imply that viruses are not easily trapped into suboptimal phenotypes and that (re)emergence is not evolutionarily constrained.

Hepatitis B escape mutants in Scottish blood donors

Hepatitis B virus (HBV) remains as the viral infection with the highest risk of transmission by transfusion. This risk is associated with window period donations, occult HBV infection (OBI) and the emergence of escape mutants, which render blood donations false negative for hepatitis B surface antigen (HBsAg) serological testing. A retrospective study was conducted to gain insights into the molecular epidemiology of HBV escape mutants in Scottish blood donors. The criterion for selection was HBV positivity either by serology or nucleic acid testing (NAT). HBsAg detection was compared across several commercial immunoassays. The full length S gene from plasma samples was PCR amplified, cloned and expressed in HepG2 cells. Eight samples showed HBsAg discordant results, while 5 OBI samples were found. Four escape mutants, containing missense mutations in the S gene, are described here. These mutations impaired HBsAg detection both from HBV infected plasma samples and from recombinant proteins derived from its infected donors. Phylogenetic analysis showed that most of the mutants were clustered in the genotype D and were closely related to strains from Asia and the Middle East. We report here a proline substitution, outside the major hydrophilic region, that impaired HBsAg detection in vivo and in vitro, warning about the risk for the emergence of vaccine escape mutants with mutations outside the major neutralisation site.

Brazilian avian metapneumovirus subtypes A and B: experimental infection of broilers and evaluation of vaccine efficacy

Avian metapneumovirus (aMPV) is a respiratory pathogen associated with the swollen head syndrome (SHS) in chickens. In Brazil, live aMPV vaccines are currently used, but subtypes A and, mainly subtype B (aMPV/A and aMPV/B) are still circulating. This study was conducted to characterize two Brazilian aMPV isolates (A and B subtypes) of chicken origin. A challenge trial to explore the replication ability of the Brazilian subtypes A and B in chickens was performed. Subsequently, virological protection provided from an aMPV/B vaccine against the same isolates was analyzed. Upon challenge experiment, it was shown by virus isolation and real time PCR that aMPV/B could be detected longer and in higher amounts than aMPV/A. For the protection study, 18 one-day-old chicks were vaccinated and challenged at 21 days of age. Using virus isolation and real time PCR, no aMPV/A was detected in the vaccinated chickens, whereas one vaccinated chicken challenged with the aMPV/B isolate was positive. The results showed that aMPV/B vaccine provided a complete heterologous virological protection, although homologous protection was not complete in one chicken. Although only one aMPV/B positive chicken was detected after homologous vaccination, replication in vaccinated animals might allow the emergence of escape mutants.

Longitudinal 2 years field study of conventional vaccination against highly pathogenic avian influenza H5N1 in layer hens

A licensed, inactivated vaccine based on a low pathogenic avian influenza virus strain (H5N2) was evaluated in layer hens kept under field conditions during a 2-year period. Vaccine efficacy was investigated by specific antibodies and by challenge-contact experiments using highly pathogenic avian influenza viruses (HPAIV) H5N1. Basic immunization with two applications induced clinical protection. Virus excretion by vaccinated hens was significantly reduced compared to non-vaccinated controls; transmission to non-vaccinated and vaccinated contact birds was not fully interrupted. Vaccination efficacy is influenced by several factors including antigenic relatedness between vaccine and field strains, but also by species, age and type of commercial uses of the host. Limitations and risks of HPAIV vaccination as silent spread of HPAIV and emergence of escape mutants must be considered a priori and appropriate corrective measures have to be installed.

Hepatitis B vaccination in children: The Taiwan experience

The world's first nationwide hepatitis B virus (HBV) universal vaccination program for infants was launched in Taiwan in July, 1984. All infants received three to four doses plasma or recombinant HBV vaccines. In addition, infants of HBeAg-positive mothers received 0.5 ml of hepatitis B immunoglobulin within 24 hours after birth. The vaccination coverage rate is as high as 97%. Seroprevalence of hepatitis B surface antigen (HBsAg) declined from 9.8% (prevaccination period) to 0.6% in children in Taipei City after 20 years of mass vaccination. The seropositive rates for HBsAg, antibody to HBsAg, and antibody to hepatitis B core antigen were 1.2%, 50.5%, and 3.7%, respectively, in those born after the vaccination program (< 20 years old) in 2004. In line with the decrease of chronic HBV infection, the incidence of hepatocellular carcinoma (HCC) also decreased in children in Taiwan. From 1981 to 1994, the incidence of HCC in 6- to 9-year-olds declined from 0.52/100,000 for those born between 1974 and 1984 to 0.13 for those born between 1984 and 1986 ( p < 0.001). We extended the observation to 2000, the incidence of HCC per 100,000 children declined from 0.54 to 0.20. The prevalence of a determinant mutants (amino acids 121–149 of HBsAg) in Taiwanese carrier children was 7.8% (eight out of 103) in 1984, increased to 19.6% (10 out of 51) in 1989, peaked at 28.1% (nince out of 32) in 1994, and remained stationary at 23.1% (three out of 13) and about 25% in 1999 and 2004, respectively; it was higher in those fully vaccinated compared with those not vaccinated. The other group of subjects who are susceptible to vaccine failure is the immunocompromized hosts. We observed some de novo HBV infection in children after liver transplantation. Despite of the success of hepatitis B immunization, childhood chronic HBV infection and HCC were not eliminated by the universal vaccination program. Among those HBsAg carriers born after the vaccination program, 89% of their mothers were found to be positive for HBsAg, indicating the importance of maternal transmission. This was also true in the mothers of children with HCC, of them 96% were HBsAg positive. After two decades of universal infant HBV vaccination, we found this program provides long-term protection for up to more than 20 years, and a universal booster is not required for the primary HBV vaccinees before adulthood. Mother-to-child transmission, although largely diminished, is still the main cause for immunoprophylaxis failure. The emergence of escape mutant did not impose increased risk of chronic infection at present. Nevertheless, development of new vaccines may overcome the vaccine failure.

RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: synthetic siRNA or vector-based shRNA?

Importance of the field: Despite the clinical benefits of highly active antiretroviral therapy (HAART), the prospect of life-long antiretroviral treatment poses significant problems, which has spurred interest in developing new drugs and strategies to treat HIV infection and eliminate persistent viral reservoirs. RNAi has emerged as a therapeutic possibility for HIV.Areas covered in this review: We discuss progress in overcoming hurdles to translating transient and stable RNAi enabling technologies to clinical application for HIV; covering the past 2 – 3 years.What the reader will gain: HIV inhibition can be achieved by transfection of chemically or enzymatically synthesized siRNAs or by DNA-based vector systems expressing short hairpin RNAs (shRNAs) that are processed intracellularly into siRNA. We compare these approaches, focusing on technical and safety issues that will guide the choice of strategy for clinical use.Take home message: Introduction of synthetic siRNA into cells or its stable endogenous production using vector-driven shRNA have been shown to suppress HIV replication in vitro and, in some instances, in vivo. Each method has advantages and limitations in terms of ease of delivery, duration of silencing, emergence of escape mutants and potential toxicity. Both appear to have potential as future therapeutics for HIV, once the technical and safety issues of each approach are overcome.

Inhibition of the Replication of Feline Iommunodeficiency Virus by Lentiviral Vector-Mediated RNA Interference in Feline Cell Lines

RNA interference (RNAi) is a sequence-specific RNA degradation process. To inhibit feline immunodeficiency virus (FIV) replication by RNAi, we generated a lentiviral vector expressing a short hairpin RNA (shRNA) that targeted the gag gene of FIV (shGag). shGag transfer significantly inhibited viral replication in cell lines that were chronically infected with FIV, i.e., the 3201/UK8 low, 3201/UK8 high, FL4, and CRFK/FIV cell lines. Moreover, 3201 cells were transduced with the lentiviral vectors and then inoculated with FIV. Although the amount of FIV proviral DNA in shGag-transduced cells was similar to that in the cells transduced with unrelated shRNA or mock-transduced cells, the amount of reverse transcriptase (RT) activity was significantly reduced in the culture supernatant of shGag-expressing cells from 15 to 27 days after inoculation. Thirty days after inoculation, no significant difference was observed in the RT activities but virus with a mutation in the target region of shGag was detected in approximately 21% of the replicated viruses. Therefore, abolishment of the silencing effect of shGag may be due to reasons other than the emergence of escape mutants. These results are useful for developing an RNAi-based gene therapy strategy for controlling FIV infection.