Non-human primate immune libraries combined with germline humanization

Panning of libraries constructed from immunised non-human primates (NHP) has not been widely used, even though this has proven to be a successful approach for the isolation of human-like antibody fragments with affinities in the nanomolar to the picomolar range. As recently demonstrated, after initial isolation of antibodies with such high affinities, germline humanization may be applied to these Fabs or scFvs to increase the similarity of their framework regions with those encoded by human germline genes. ‘Germlinized’ antibody fragments may be converted to full size IgGs; indications are given that these IgGs could be better tolerated in clinical use than human antibodies. The use of the combination of NHP immune libraries and germline humanization thus may compete with use of libraries of human origin, whether naïve or immune, and with synthetic libraries. In this report, the various approaches will be compared, and advantages of the two-step NHP-based method, as well as corresponding intellectual property aspects, will be discussed.     

A novel platform to produce human monoclonal antibodies: The next generation of therapeutic human monoclonal antibodies discovery

A new technology has been developed by immunologix that allows human antibodies to be quickly generated against virtually any antigen. Using a novel process, naïve human B cells are isolated from tonsil tissue and transformed with efficiency up to 85%, thus utilizing a large portion of the human VDJ/VJ repertoire. Through ex vivo stimulation, the B cells class switch and may undergo somatic hypermutation, thus producing a human “library” of different IgG antibodies that can then be screened against any antigen. Since diversity is generated ex vivo, sampling immunized or previously exposed individuals is not necessary. Cells producing the antibody of interest can be isolated through limiting dilution cloning and the human antibody from the cells can be tested for biological activity. No humanization is necessary because the antibodies are produced from human B cells. By eliminating immunization and humanization steps and screening a broadly diverse library, this platform should reduce both the cost and time involved in producing therapeutic monoclonal antibody candidates.Key words: human, antibody, monoclonal, novel platform, naïve, B cell, therapeutic     

Development of a novel mammalian display system for selection of antibodies against membrane proteins

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 10⁹ variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (K_D) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.     

Mining human antibody repertoires

Human monoclonal antibodies (mAbs) have become drugs of choice for the management of an increasing number of human diseases. Human antibody repertoires provide a rich source for human mAbs. Here we review the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. In particular, we discuss the selection of human mAbs from naïve, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display. Our reliance on different strategies is shifting as we gain experience and refine methods to the efficient generation of human mAbs with superior pharmacokinetic and pharmacodynamic properties.Key words: human monoclonal antibodies, B cells, hybridoma technology, display technologies, antibody libraries, antibody engineering     

Global Analysis Reveals Families of Chemical Motifs Enriched for hERG Inhibitors

Promiscuous inhibition of the human ether-à-go-go-related gene (hERG) potassium channel by drugs poses a major risk for life threatening arrhythmia and costly drug withdrawals. Current knowledge of this phenomenon is derived from a limited number of known drugs and tool compounds. However, in a diverse, naïve chemical library, it remains unclear which and to what degree chemical motifs or scaffolds might be enriched for hERG inhibition. Here we report electrophysiology measurements of hERG inhibition and computational analyses of >300,000 diverse small molecules. We identify chemical ‘communities’ with high hERG liability, containing both canonical scaffolds and structurally distinctive molecules. These data enable the development of more effective classifiers to computationally assess hERG risk. The resultant predictive models now accurately classify naïve compound libraries for tendency of hERG inhibition. Together these results provide a more complete reference map of characteristic chemical motifs for hERG liability and advance a systematic approach to rank chemical collections for cardiotoxicity risk.     

Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes

Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.     

Discussions on the quality of antibodies are no reason to ban animal immunization

Debates about the source of antibodies and their use are confusing two different issues. A ban on life immunization would have no repercussions on the quality of antibodies. Subject Categories: S&S: Economics & Business, Methods & Resources, Chemical Biology

There is an ongoing debate on how antibodies are being generated, produced and used (Gray, 2020; Marx, 2020). Or rather, there are two debates, which are not necessarily related to each other. The first one concerns the quality of antibodies used in scientific research and the repercussions for the validity of results (Bradbury & Pluckthun, 2015). The second debate is about the use of animals to generate and produce antibodies. Although these are two different issues, we observe that the debates have become entangled with arguments for one topic incorrectly being used to motivate the other and vice versa. This is not helpful, and we should disentangle the knot.Polyclonal antibodies are being criticized because they suffer from cross‐reactivity, high background and batch‐to‐batch variation (Bradbury & Pluckthun, 2015). Monoclonal antibodies produced from hybridomas are criticized because they often lack specificity owing to genetic heterogeneity introduced during hybridoma generation that impairs the quality of the monoclonals (Bradbury et al, 2018). These are valid criticisms and producing antibodies in a recombinant manner will, indeed, help to improve quality and specificity. But a mediocre antibody will remain a mediocre antibody, no matter how it is produced. Recombinant methods will just produce a mediocre antibody more consistently.Getting a good antibody is not easy and much depends on the nature and complexity of the antigen. And low‐quality antibodies are often the result of poor screening, poor quality control, incomplete characterization and the lack of international standards. Nevertheless, the technologies to ensure good selection and to guarantee consistent quality are much more advanced than a decade ago, and scientists and antibody producers should implement these to deliver high‐quality antibodies. Whether antibodies are generated by animal immunization or from naïve or synthetic antibody libraries is less relevant; they can all be produced recombinantly, and screening and characterization are needed in all cases to determine quality, and if the antibody is fit for purpose.But criticisms on the quality of many antibodies and pleas for switching to recombinant production of antibodies cannot be mixed up with a call to ban animal immunization. The EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) recently published a recommendation to stop using animals for generating and producing antibodies for scientific, diagnostic and even therapeutic applications (EURL ECVAM, 2020). This recommendation is mainly supported by scientists who seem to be biased towards synthetic antibody technology for various reasons. Their main argument is that antibodies derived from naïve or synthetic libraries are a valid (and exclusive) alternative. But are they?One can certainly select antibodies from non‐immune libraries, and, depending on the antigen and the type of application, these antibodies can be fit for purpose. In fact, a few of such antibodies have made it to the market as therapeutics, Adalimumab (Humira®) being a well‐known example. But up to now, the vast majority of antibodies continues to come from animal immunization (Lu et al, 2020). And there is a good reason for that. It is generally possible to generate a few positive hits in a naïve/synthetic library; and the more diverse the library, the more hits one is likely to get. But many decades of experience with immunization of animals—especially when they are outbred—shows that they generate larger amounts of antibodies with superior properties. And the more complex your antigen is, the more the balance swings towards animal immunization if you want to have a guarantee for success.There are different factors at work here. First, the immune system of mammals has evolved over millions of years to efficiently produce excellent antibodies against a very diverse range of antigens. Second, presenting the antigen multiple times in its desired (native) conformation to the animal immune system exploits the natural maturation process to fine‐tune the immune response against particular qualities. Another factor is that in vivo maturation seems to select against negative properties such as self‐recognition and aggregation. It also helps to select for important properties that go beyond mere molecular recognition (Jain et al, 2017). In industrial parlance, antibodies from animal immunization are more “developable” and have favourable biophysical properties (Lonberg, 2005). Indeed, the failure rate for antibodies selected from naïve or synthetic libraries is significantly higher.Of course, the properties of synthetic antibodies selected from non‐immune libraries can be further matured in vitro, for example by light chain shuffling or targeted mutagenesis of the complementarity determining region (CDR). While this method has become more sophisticated over the years, it remains a very complex and iterative process without guarantee that it produces a high‐quality antibody.Antibodies are an ever more important tool in scientific research and a growing area in human and veterinary therapeutics. Major therapeutic breakthroughs in immunology and oncology in the past decades are based on antibodies (Lu et al, 2020). The vast majority of these therapeutic antibodies were derived from animals. An identical picture appears when you look at the antibodies in fast‐track development to combat the current COVID‐19 crisis: again, the vast majority are either derived from patients or from animal immunizations. The same holds true for antibodies that are used in diagnostics and epidemiologic studies for COVID‐19.It is for that reason that we need the tools and methods that guarantee antibodies of the highest quality and provide the best chance for success. The COVID‐19 pandemic is only one illustration of this need. If we block access to these tools, both scientific research and society at large will be negatively impacted. We therefore should not limit ourselves to naïve and synthetic libraries. Animal immunization remains an inevitable method that needs to stay. But we all agree that these immunizations must be performed under best practice to further reduce the harm to animals.     

Accumulation of Self-Reactive Na?ve and Memory B Cell Reveals Sequential Defects in B Cell Tolerance Checkpoints in Sj?gren’s Syndrome

Sjögren’s syndrome (SS) is an autoimmune disease characterised by breach of self-tolerance towards nuclear antigens resulting in high affinity circulating autoantibodies. Although peripheral B cell disturbances have been described in SS, with predominance of naïve and reduction of memory B cells, the stage at which errors in B cell tolerance checkpoints accumulate in SS is unknown. Here we determined the frequency of self- and poly-reactive B cells in the circulating naïve and memory compartment of SS patients. Single CD27−IgD+ naïve, CD27+IgD+ memory unswitched and CD27+IgD− memory switched B cells were sorted by FACS from the peripheral blood of 7 SS patients. To detect the frequency of polyreactive and autoreactive clones, paired Ig V_H and V_L genes were amplified, cloned and expressed as recombinant monoclonal antibodies (rmAbs) displaying identical specificity of the original B cells. IgV_H and V_L gene usage and immunoreactivity of SS rmAbs were compared with those obtained from healthy donors (HD). From a total of 353 V_H and 293 V_L individual sequences, we obtained 114 rmAbs from circulating naïve (n = 66) and memory (n = 48) B cells of SS patients. Analysis of the Ig V gene repertoire did not show significant differences in SS vs. HD B cells. In SS patients, circulating naïve B cells (with germline V_H and V_L genes) displayed a significant accumulation of clones autoreactive against Hep-2 cells compared to HD (43.1% vs. 25%). Moreover, we demonstrated a progressive increase in the frequency of circulating anti-nuclear naïve (9.3%), memory unswitched (22.2%) and memory switched (27.3%) B cells in SS patients. Overall, these data provide novel evidence supporting the existence of both early and late defects in B cell tolerance checkpoints in patients with SS resulting in the accumulation of autoreactive naïve and memory B cells.     

CD4+ T cells in aged or thymectomized recipients of allogeneic stem cell transplantations

Background

CD4+CD25highFOXP3+ regulatory T (Treg) cells, which include thymus-derived and peripherally induced cells, play a central role in immune regulation, and are therefore crucial to prevent graft-versus-host disease (GVHD). The increasing use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for elderly patients with thymus regression, and our case of allo-HSCT shortly after total thymectomy, raised questions about the activity of thymus-derived Treg cells and peripherally induced Treg cells, which are otherwise indistinguishable.

Results

We found that despite pre-transplant thymectomy or older age, both naïve and effector Treg cells, as well as naïve and effector conventional T cells, proliferated in allo-HSCT recipients. Higher proportions of total Treg cells 1 month post allo-HSCT, and naïve Treg cells 1 year post allo-HSCT, appeared in patients achieving complete chimera without developing significant chronic GVHD, including our thymectomized patient, compared with patients who developed chronic GVHD.

Conclusions

Treg cells that modulate human allogeneic immunity may arise peripherally as well as in the thymus of allo-HSCT recipients.     

An AGM Model for Changes in Complement during Pregnancy: Neutralization of Influenza Virus by Serum Is Diminished in Late Third Trimester

Pregnant women in the third trimester are at increased risk of severe influenza disease relative to the general population, though mechanisms behind this are not completely understood. The immune response to influenza infection employs both complement (C′) and antibody (Ab). The relative contributions of these components to the anti-viral response are difficult to dissect because most humans have pre-existing influenza-specific Abs. We developed the African green monkey (AGM) as a tractable nonhuman primate model to study changes in systemic innate immunity to influenza during pregnancy. Because the AGMs were influenza-naïve, we were able to examine the role of C′ in influenza virus neutralization using serum from non-pregnant animals before and after influenza infection. We determined that serum from naïve AGMs neutralized influenza via C′, while post-infection neutralization did not require C′, suggesting an Ab-mediated mechanism. The latter mimicked neutralization using human serum. Further, we found that ex vivo neutralization of influenza with both naïve and influenza-immune AGM serum occurred by virus particle aggregation and lysis, with immune serum lysing virus at a much higher rate than naïve serum. We hypothesized that the anti-influenza C′ response would diminish late in AGM pregnancy, corresponding with the time when pregnant women suffer increased influenza severity. We found that influenza neutralization capacity is significantly diminished in serum collected late in the third trimester. Strikingly, we found that circulating levels of C3, C3a, and C4 are diminished late in gestation relative to nonpregnant animals, and while neutralization capacity and serum C3a return to normal shortly after parturition, C3 and C4 levels do not. This AGM model system will enable further studies of the role of physiologic and hormonal changes in downregulating C′-mediated anti-viral immunity during pregnancy, and it will permit the identification of therapeutic targets to improve outcomes of influenza virus infection in pregnant women.     

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.     

Foreign DNA acquisition by the I-F?CRISPR–Cas system requires all components of the interference machinery

CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR–Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR–Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR–Cas systems.     

Rapid Proliferation and Differentiation of a Subset of Circulating IgM Memory B Cells to a CpG/Cytokine Stimulus In Vitro

Circulating human IgM expressing memory B cells have been incompletely characterized. Here, we compared the phenotype and in vitro functional response (capacity to proliferate and differentiate to antibody secreting cells) in response to CpG and a cytokine cocktail (IL-2, IL-6, and IL-10) of sorted naïve B cells, IgM memory B cells and isotype-switched circulating memory B cells. Compared to naïve B cells, IgM memory B cells had lower integrated mean fluorescence intensity (iMFI) of BAFF-R, CD38, CD73, and IL-21R, but higher iMFI of CD95, CD11c, TLR9, PD-1, and CD122. Compared to switched memory B cells, IgM memory B cells had higher iMFI of BAFF-R, PD-1, IL-21R, TLR9, and CD122, but lower iMFI of CD38, CD95, and CD73. Four days after receiving the CpG/cytokine cocktail, higher frequencies of IgM than switched memory B cells—and these in turn greater than naïve cells—proliferated and differentiated to antibody secreting cells. At this time point, a small percentage (median of 7.6%) of stimulated IgM memory B cells changed isotype to IgG. Thus, among the heterogeneous population of human circulating IgM memory B cells a subset is capable of a rapid functional response to a CpG/cytokine stimulus in vitro.     

Prior Infection of Chickens with H1N1 or H1N2 Avian Influenza Elicits Partial Heterologous Protection against Highly Pathogenic H5N1

There is a critical need to have vaccines that can protect against emerging pandemic influenza viruses. Commonly used influenza vaccines are killed whole virus that protect against homologous and not heterologous virus. Using chickens we have explored the possibility of using live low pathogenic avian influenza (LPAI) A/goose/AB/223/2005 H1N1 or A/WBS/MB/325/2006 H1N2 to induce immunity against heterologous highly pathogenic avian influenza (HPAI) A/chicken/Vietnam/14/2005 H5N1. H1N1 and H1N2 replicated in chickens but did not cause clinical disease. Following infection, chickens developed nucleoprotein and H1 specific antibodies, and reduced H5N1 plaque size in vitro in the absence of H5 neutralizing antibodies at 21 days post infection (DPI). In addition, heterologous cell mediated immunity (CMI) was demonstrated by antigen-specific proliferation and IFN-γ secretion in PBMCs re-stimulated with H5N1 antigen. Following H5N1 challenge of both pre-infected and naïve controls chickens housed together, all naïve chickens developed acute disease and died while H1N1 or H1N2 pre-infected chickens had reduced clinical disease and 70–80% survived. H1N1 or H1N2 pre-infected chickens were also challenged with H5N1 and naïve chickens placed in the same room one day later. All pre-infected birds were protected from H5N1 challenge but shed infectious virus to naïve contact chickens. However, disease onset, severity and mortality was reduced and delayed in the naïve contacts compared to directly inoculated naïve controls. These results indicate that prior infection with LPAI virus can generate heterologous protection against HPAI H5N1 in the absence of specific H5 antibody.     

Evaluation of the Murine Immune Response to Xenopsylla cheopis Flea Saliva and Its Effect on Transmission of Yersinia pestis

Background/Aims

Arthropod-borne pathogens are transmitted into a unique intradermal microenvironment that includes the saliva of their vectors. Immunomodulatory factors in the saliva can enhance infectivity; however, in some cases the immune response that develops to saliva from prior uninfected bites can inhibit infectivity. Most rodent reservoirs of Yersinia pestis experience fleabites regularly, but the effect this has on the dynamics of flea-borne transmission of plague has never been investigated. We examined the innate and acquired immune response of mice to bites of Xenopsylla cheopis and its effects on Y. pestis transmission and disease progression in both naïve mice and mice chronically exposed to flea bites.

Methods/Principal Findings

The immune response of C57BL/6 mice to uninfected flea bites was characterized by flow cytometry, histology, and antibody detection methods. In naïve mice, flea bites induced mild inflammation with limited recruitment of neutrophils and macrophages to the bite site. Infectivity and host response in naïve mice exposed to flea bites followed immediately by intradermal injection of Y. pestis did not differ from that of mice infected with Y. pestis without prior flea feeding. With prolonged exposure, an IgG1 antibody response primarily directed to the predominant component of flea saliva, a family of 36–45 kDa phosphatase-like proteins, occurred in both laboratory mice and wild rats naturally exposed to X. cheopis, but a hypersensitivity response never developed. The incidence and progression of terminal plague following challenge by infective blocked fleas were equivalent in naïve mice and mice sensitized to flea saliva by repeated exposure to flea bites over a 10-week period.

Conclusions

Unlike what is observed with many other blood-feeding arthropods, the murine immune response to X. cheopis saliva is mild and continued exposure to flea bites leads more to tolerance than to hypersensitivity. The immune response to flea saliva had no detectable effect on Y. pestis transmission or plague pathogenesis in mice.     

By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.

By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection. Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage. Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues. The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA). Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range. The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3. However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin. Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro.     

Timing of Immune Escape Linked to Success or Failure of Vaccination

Successful vaccination against HIV should limit viral replication sufficiently to prevent the emergence of viral immune escape mutations. Broadly directed immunity is likely to be required to limit opportunities for immune escape variants to flourish. We studied the emergence of an SIV Gag cytotoxic T cell immune escape variant in pigtail macaques expressing the Mane-A*10 MHC I allele using a quantitative RT-PCR to measure viral loads of escape and wild type variants. Animals receiving whole Gag expressing vaccines completely controlled an SIV_mac251 challenge, had broader CTL responses and exhibited minimal CTL escape. In contrast, animals vaccinated with only a single CTL epitope and challenged with the same SIV_mac251 stock had high levels of viral replication and rapid CTL escape. Unvaccinated naïve animals exhibited a slower emergence of immune escape variants. Thus narrowly directed vaccination against a single epitope resulted in rapid immune escape and viral levels equivalent to that of naïve unvaccinated animals. These results emphasize the importance of inducing broadly directed HIV-specific immunity that effectively quashes early viral replication and limits the generation of immune escape variants. This has important implications for the selection of HIV vaccines for expanded human trials.