Summary Plant molecular farming (PMF) is rapidly gaining traction as a viable alternative to the currently accepted paradigm of producing biologics. While the platform is potentially cheaper and more scalable than conventional manufacturing systems, expression yields and appropriate post‐translational modifications along the plant secretory pathway remain a challenge for certain proteins. Viral fusion glycoproteins in particular are often expressed at low yields in plants and, in some cases, may not be appropriately processed. Recently, however, transiently or stably engineering the host plant has shown promise as a strategy for producing heterologous proteins with more complex maturation requirements. In this study we investigated the co‐expression of a suite of human chaperones to improve the production of a human immunodeficiency virus (HIV) type 1 soluble gp140 vaccine candidate in Nicotiana benthamiana plants. The co‐expression of calreticulin (CRT) resulted in a dramatic increase in Env expression and ameliorated the endoplasmic reticulum (ER) stress response ‐ as evidenced by lower transcript abundance of representative stress‐responsive genes. The co‐expression of CRT similarly improved accumulation of glycoproteins from Epstein‐Barr virus (EBV), Rift Valley fever virus (RVFV) and chikungunya virus (CHIKV), suggesting that the endogenous chaperone machinery may impose a bottleneck for their production. We subsequently successfully combined the co‐expression of human CRT with the transient expression of human furin, to enable the production of an appropriately cleaved HIV gp140 antigen. These transient plant host engineering strategies are a promising approach for the production of high yields of appropriately processed and cleaved viral glycoproteins.

SummaryPlant molecular farming offers a cost‐effective and scalable approach to the expression of recombinant proteins which has been proposed as an alternative to conventional production platforms for developing countries. In recent years, numerous proofs of concept have established that plants can produce biologically active recombinant proteins and immunologically relevant vaccine antigens that are comparable to those made in conventional expression systems. Driving many of these advances is the remarkable plasticity of the plant proteome which enables extensive engineering of the host cell, as well as the development of improved expression vectors facilitating higher levels of protein production. To date, the only plant‐derived viral glycoprotein to be tested in humans is the influenza haemagglutinin which expresses at ~50 mg/kg. However, many other viral glycoproteins that have potential as vaccine immunogens only accumulate at low levels in planta. A critical consideration for the production of many of these proteins in heterologous expression systems is the complexity of post‐translational modifications, such as control of folding, glycosylation and disulphide bridging, which is required to reproduce the native glycoprotein structure. In this review, we will address potential shortcomings of plant expression systems and discuss strategies to optimally exploit the technology for the production of immunologically relevant and structurally authentic glycoproteins for use as vaccine immunogens.

In this study we examined potential associations of HPV infection with the cervical microbiota. Cervical samples were collected from 87 HIV-seronegative reproductive-age Black South African women. Microbiota were characterized by Illumina sequencing of the V3-V4 hypervariable regions of the bacterial 16S rRNA gene. Thirty seven (42.5%) and 30 (34.5%) of the women had prevalent HPV and high-risk (HR)-HPV, respectively. Only 23 women (26.4%) had cervical microbiota dominated by a single Lactobacillus species ( L. crispatus (2/87 (2.3%)), L. jensenii (2/87 (2.3%)), and L. iners (19/87 (21.8%)). The majority of the women (56/87 (64.4%)) had diverse cervical microbiota consisting of mainly bacterial vaginosis-associated bacteria. The remaining women (8/87 (9.2%)) had microbiota dominated by Aerococcus , Streptococcus , Chlamydia or Corynebacterium. Women with HR-HPV had significantly higher relative abundances of Aerococcaceae , Pseudomonadaceae and Bifidobacteriaceae compared to those with low-risk (LR)-HPV or no HPV-infection (LDA score >2.0, p < 0.05, q < 0.2). Gardnerella, Sneathia, and Atopobium were also found at greater relative abundances in HR-HPV-infected women compared to those with low-risk (LR)-HPV or no HPV-infection (LDA score >2.0, p < 0.05), although the difference was not significant after FDR-adjustment (q > 0.2). Further investigations of the bacterial taxa significantly enriched in HR-HPV-infected women are warranted.

The development of effective vaccines is urgently needed to curb the spread of human immunodeficiency virus type 1 (HIV-1). A major focal point of current HIV vaccine research is the production of soluble envelope (Env) glycoproteins which reproduce the structure of the native gp160 trimer. These antigens are produced in mammalian cells, which requires a sophisticated infrastructure for manufacture that is mostly absent in developing countries. The production of recombinant proteins in plants is an attractive alternative for the potentially cheap and scalable production of vaccine antigens, especially for developing countries. In this study, we developed a transient expression system in Nicotiana benthamiana for the production of soluble HIV Env gp140 antigens based on two rationally selected virus isolates (CAP256 SU and Du151). The scalability of the platform was demonstrated and both affinity and size exclusion chromatography (SEC) were explored for recovery of the recombinant antigens. Rabbits immunized with lectin affinity-purified antigens developed high titres of binding antibodies, including against the V1V2 loop region, and neutralizing antibodies against Tier 1 viruses. The removal of aggregated Env species by gel filtration resulted in the elicitation of superior binding and neutralizing antibodies. Furthermore, a heterologous prime-boost regimen employing a recombinant modified vaccinia Ankara (rMVA) vaccine, followed by boosts with the SEC-purified protein, significantly improved the immunogenicity. To our knowledge, this is the first study to assess the immunogenicity of a near-full length plant-derived Env vaccine immunogen.