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Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally., (© 2021. The Author(s).)

Background: Positive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance., Results: Using influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3' ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor (NAI) resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays., Conclusion: This approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.

In the event of an influenza pandemic, the use of oseltamivir (OTV) will undoubtedly increase and therefore it is more likely that OTV-resistant influenza strains will also arise. OTV-resistance genotyping using sequence-based testing on viruses isolated in cell culture is time consuming and less likely to detect the low-level presence of drug-resistant virus populations. We have developed a novel rolling circle amplification (RCA) method to achieve the sensitive detection of OTV-resistant viruses from clinical specimens. Using artificially created templates, RCA could detect the presence of OTV-resistant mutations (N2: 119V, 292K, N1: 274Y) even if the population carrying the mutations was <1% of the total. By applying RCA to clinical samples, we identified the emergence of the 274Y mutation in one OTV-treated patient, as well as in seven individuals who were treatment-naïve (confirming community transmission of 274Y-containing resistant influenza A H1N1). These results were further confirmed by neuraminidase region sequencing. In conclusion, RCA technology can provide rapid (<24 h), high-throughput diagnosis of OTV resistance mutations with a high specificity and sensitivity.

Primary or transmitted antiretroviral drug resistance mutations pose a significant obstacle for optimizing antiviral treatment. When present at low-levels, resistance mutations are less likely to be detected by standard genotyping assays. This study utilizes a novel rolling circle amplification (RCA) method using padlock probes to achieve the sensitive, specific and low-level detection of the NNRTI resistance K103N from 59 HIV+ treatment-naïve patients from Beijing, China. Using standard genotyping methods, primary drug resistance mutations to either protease or RT inhibitors were found in 25% (15/59) of patients attending hospital clinics in Beijing. Among these 15 patients with antiretroviral (ARV) resistance mutations, standard sequence-based genotyping revealed that most (10/15) had the 103N. Using a highly sensitive RCA assay, 5 more patients among the 59 treatment-naïve cohort were found to have the 103N, but at low-levels, leading to an overall rate of 103N at 25.4% (15/59) in this population. The high prevalence of the 103N suggests that baseline resistance testing should be performed before treatment in this population. Importantly, the new RCA technology allows large-scale, sensitive detection of drug resistance mutations, including detection of minority populations with minimal equipment requirement.

Objective: To develop a method for single syncytia isolation and delineate the possible contribution of syncytia to intersubtype recombination., Design: We dually infected whole peripheral mononuclear blood cells with subtype A and D viruses and studied syncytia in vitro and developed a method to isolate individual syncytia to further study HIV variants/dual infections, viral isolation, proviral copies in single syncytia and possible intersubtype recombination in dual cultures containing syncytia using real time PCR., Methods: Cell culture-based single syncytia isolation, PCR and cloning to determine the nature of HIV variants and real-time PCR to determine proviral copies per individual syncytium and intersubtype recombination in dual cultures. Viral coculture from single syncytia and p24 antigen determination for assessing viral replication in vitro., Results: Our results show the feasibility that not only can single syncytia be successfully isolated, but the viruses from individual syncytia can also be grown in vitro. They also demonstrate the ability of single syncytia to bring diverse HIV-1 subtypes together along with the possible contribution to intersubtype recombination in vitro. Up to 40% of single syncytia harbored both input HIV-1 subtypes and single syncytium could harbor as many as 2000 proviral DNA copies, which exceeds the limit seen in a single cell., Conclusion: These analyses are unique in experimentally confirming the previously held belief that single syncytia can harbor multiple HIV strains and that they can serve as a breeding ground for heterozygous virions and this may contribute toward viral diversity and intersubtype recombination.

The HIV protease-reverse transcriptase (PR-RT) (1047 bp), gp120-env (891 bp) and gp41-env (547 bp) regions from the plasma of 115 HIV-1-infected patients in Kuala Lumpur (KL), Malaysia were sequenced. Detailed phylogenetic and bootscanning analyses were performed to determine the mosaic structure of the HIV-1 strains and their recombination breakpoint(s). Among the 50 patient samples in which all three regions could be amplified, the HIV-1 CRF01&#95;AE subtype (46%) was predominant followed by subtypes B (10%) and B' (6%). A total of 9/50 (18%) patients were infected with a CRF01&#95;AE/B inter-subtype recombinant, displaying a recombinant form (RF)(PR-RT), CRF01&#95;AE(gp120-env) and CRF01&#95;AE(gp41-env). This RF was derived from the Thai variants of CRF01&#95;AE and B' subtype, with two distinct B' subtype segments in the backbone of CRF01&#95;AE, similar to the newly identified CRF33&#95;01B. In addition, one sample demonstrated a close structural relationship with the new CRF33&#95;01B in the PR-RT region but displayed B' segment in part of the env region (RF(PR-RT), CRF01&#95;AE/B'(gp120-env) and B'(gp41-env)) indicating continuing evolution of CRF33&#95;01B. The remaining 18% of samples were identified as unique recombinant forms (URFs).

Background: HIV-1 prevalence in Kenya among women aged between 15-19 years is approximately 23%. These women are prospective mothers and therefore can play an important role in mother-to-child transmission of HIV. The risk of a seropositve mother transmitting the virus to her infant is 25-35% in developing countries, such as Kenya, where antiretroviral drugs are not readily available., Objectives: This study was undertaken to evaluate the molecular nature of HIV-1 strains, assess recombination and it's relevance in mother-to-child transmission in Kenya., Study Design: HIV-1 gag and gp120 sequences were derived from peripheral blood mononuclear cells (PBMC) of 16 infected mothers and infants, from Kisumu, Kenya. PCR, cloning, and phylogenetic analyses were conducted to examine any sequence differences between HIV-1 strains derived from mother-infant pairs., Results: The cohort consisted of seven pairs harboring possible subtype A/D recombinants, eight pairs with apparent pure A or D strains and one possible dual infection. This dual infection comprised of a pure subtype A region and an A/D recombinant, and was detected in one of the mother's sample. Interestingly, only the recombinant virus was detected in the paired baby sample., Conclusions: This study shows that HIV-1 inter-subtype recombinants can be effectively transmitted vertically to infants, and could possibly be favored in this setting where multiple subtypes infect women. Together, dual infections and the co-existence of multiple HIV-1 subtypes is encouraging the emergence of recombinant HIV strains and their rapid dispersal.

As vertical transmission of HIV-1 is an ongoing problem in East Africa, we analyzed HIV-1 strains of infected mothers, from Kisumu, Kenya. We sequenced the gag and gp120 regions from peripheral blood mononuclear cells (PBMC) of 15 HIV-infected mothers attending an antenatal clinic. PCR, cloning, bootscanning, using the program Simplot, and phylogenetic analyses were conducted to assign subtypes and identify recombinants. Our analyses showed two dual infections from patients who had infections with pure subtypes and recombinants subtype D. In addition, we also noted the presence of subsubtype A1 and A2, as well as unique recombinants in this area. These results imply that the HIV epidemic in western Kenya is a dynamic one and is continually evolving. Therefore, continued monitoring of the epidemic in this region is necessary if a vaccine for the area is to be developed.

As the human immunodeficiency virus (HIV) pandemic progresses, an increasing number of recombinant viruses have been identified and in many geographical regions they are now the predominating strain. These recombinants are formed when an individual has acquired a co-infection or superinfection with more than one HIV-1 strain or subtype. Thus, dually infected individuals provide opportunities for studying HIV recombinants and viral interactions between infecting strains in vivo. The possible epidemiological, clinical and therapeutic implications of dual infections and recombination are many. Recombination may result in the emergence of more pathogenic and virulent HIV strains with altered fitness, tropism, and resistance to multiple drugs, and may hamper the development of subtype-based vaccines. This review is aimed at providing a more thorough understanding of dual infections (both co-infection and super-infection) and the possible consequences of the emergence of recombinant HIV-1 strains.