Different Modes of Retrovirus Restriction by Human APOBEC3A and APOBEC3G In Vivo

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.United States. Public Health Service (Grant R01-AI-085015)United States. Public Health Service (Grant T32-CA115299 )United States. Public Health Service (Grant F32-AI100512

Deamination of M-MLV viral DNA and RNA in A3G transgenic mice.

<p>Splenic DNA or RNA from virions was isolated from the infected mice described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004145#ppat-1004145-g003" target="_blank">Figure 3</a> and cloned and sequenced. In most cases > 10 sequences from 4 different mice were analyzed, as indicated in the figure. For the viral RNA samples, the viruses from 5 animals were pooled for sequencing. Shown are the G to A changes in the sequences; other mutations are indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004145#ppat-1004145-t001" target="_blank">Table 1</a>. Red  =  GG > AG, cyan  =  GA > AA, green  =  GC > AC and magenta  =  GT > AT transitions. Red arrows denote mutation hotspots seen in viruses isolated from A3G^high and A3G^low mice; black arrows denote hotspots identified only in A3G^high mice.</p

Expression of A3A and A3G in BMDCs restrict incoming murine retroviruses.

<p>A) Infection of BMDCs with M-MLV. RT-qPCR analysis of genomic DNA with M-MLV-specific primers, normalized to GAPDH. B) MMTV infection of BMDCs with MMTV. RT-qPCR analysis of genomic DNA with MMTV-specific primers, normalized to GAPDH. Shown are the results of 2 independent experiments (open bars, expt.1; closed bars, expt. 2) with 3 technical replicates in each experiment. Error bars (standard deviation) and p values were calculated for each experiment. *, p≤.01 based on one-way ANOVA.</p

A3A and A3G restrict murine retrovirus infection <i>in vivo</i>.

<p>A) Newborn mice were infected with M-MLV and 16 days post-infection, virus titers in spleens were measured. Each point represents the titer obtained from an individual mouse; the average for each group is shown by a horizontal bar. The transgenic mice were derived from 2–3 litters each; the knockout mice are the littermates of the transgenic mice. N = 12 A3G^high, 6 A3G^low, 10 A3A^high, 7 A3A^low, 42 KO and 6 WT mice. B) Five day old mice were infected with MMTV and 3 weeks post-infection, DNA was isolated from spleens and subjected to RT-qPCR with MMTV-specific primers. Each point represents the proviral DNA levels measured in splenic DNA from an individual mouse; the average for each group is shown by a horizontal bar. The transgenic mice were derived from 2–3 litters each; the knockout mice are the littermates of the transgenic mice. N = 5 A3G^high, 10 A3G^low, 12 A3A^high, 6 A3A^low and 24 KO mice. *, p≤0.0001, **, p≤.001, ***, p≤.01, NS, not significant (Mann-Whitney <i>t</i> test).</p

Mutation frequencies in MLV and MMTV proviruses in splenic DNA of infected A3A and A3G mice.

<p>Analysis was performed on the cloned DNA represented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004145#ppat-1004145-g004" target="_blank">Figure 4</a> (3–4 mice/group; 10–15 sequences/mouse). Unique clones were distinguished by their mutation pattern. M-MLV has a total of 47 GG and 33 GA motifs in the 549bp target sequence, MMTV has 34 GGs and 56 GAs in 673bp and F-MLV has 58 GG and 29 GA motifs in 586bp.</p

Vif counteracts A3G in transgenic mice.

<p>Newborn mice were infected with F-MLV-2A or F-MLV-2A-vif. A) Splenic extracts and isolated virions were analyzed by western blot. A3G was detected with anti-A3G antisera. Shown is a representative western blot from individual mice (KO and A3G^high) infected with F-MLV-2A and F-MLV-2A-vif. This experiment was repeated twice with 1 additional mouse of each genotype and gave similar results. B) Virus titers in spleens were measured. Each point represents the titer obtained from an individual mouse; the average for each group is shown by a horizontal bar. The transgenic mice were derived from 1–3 litters each; the knockout mice are the littermates of the transgenic mice. N = 11 KO, 4 A3G^high and 5 A3G^low mice infected with F-MLV-2A-vif and 12 KO, 4 A3G^high and 4 A3G^low mice infected with FMLV-2A. *, p≤0.02; **, p≤0.004; NS, not significant (Mann-Whitney T test).</p

Expression of A3A and A3G transgenes.

<p>A) RT-qPCR analysis of RNA isolated from different tissues of the A3G^high and A3G^low strains. B) RT-qPCR analysis of RNA isolated from different tissues of the A3A^high and A3A^low strains. Shown for comparison for both graphs are the endogenous A3 levels in nontransgenic C57BL/6 mice (mA3), as well as A3A and A3G expression in human H9 cells and human PBMCs (average of 2 individuals). The mice used for this analysis were uninfected. Both panels are representative of 2 independent experiments with a different mouse of each genotype. Error bars denote standard deviation of technical replicates.</p

Cellular lysates from splenocytes derived from uninfected transgenic, wild type (BL/6) or mA3 knockout (KO) mice or from 293T cell lines over-expressing A3A or A3G were incubated with a 3′-fluorophore labeled 50-mer single-stranded oligonucleotide (S50) containing cytosine in the sequence context preferred by A3G (S50-CCC) or A3A (S50-TTC).

<p>Deamination was detected by uracil excision by UDG followed by fragmentation of the resulting abasic site by NaOH and heat, resulting in a 35-mer product (P35). High levels of activity in the 293/A3A samples result in deamination at multiple potential cytosines in the S50-CCC substrate (P37). This experiment was performed several times with the same lysates, with similar results.</p

Open-Label Pilot Study of Genetically Engineered NY-ESO-1 Specific T Cells (GSK3377794) Alone or in Combination with Pembrolizumab in Relapsed and Refractory Multiple Myeloma

Background: Emerging clinical data demonstrate that adoptive cellular therapy has potential to be practice-changing in the management of relapsed/refractory multiple myeloma (MM). NY-ESO-1 TCR T (GSK3377794) are autologous polyclonal T cells transduced by a self-inactivating lentiviral vector to express an affinity-enhanced TCR capable of recognizing NY-ESO-1 or LAGE-1a antigenic peptides in complex with HLA-A2. In prior studies, encouraging clinical activity has been observed with GSK3377794 treatment in patients with synovial sarcoma, melanoma, myxoid/round cell liposarcoma, and in MM patients receiving GSK3377794 after autologous stem cell transplant (ASCT). NY-ESO-1 and LAGE-1a are immunogenic cancer/testis antigens frequently overexpressed in MM and are linked to poor clinical outcome. While a number of phase 1 and 2 trials are evaluating GSK3377794 in solid tumors, this abstract presents a trial in progress aiming to evaluate safety and efficacy of GSK3377794 alone or in combination with the anti-PD1 inhibitor, pembrolizumab, in patients with MM. PD-1 expression on CD8 T cells has been observed in MM patients previously treated with GSK3377794 and can limit adaptive immune response. This has also been described as a mechanism of resistance and relapse in CD19 CAR T-cell trials (Fraietta et al, Nat Med 2018). Thus, we hypothesize that combining GSK3377794 and pembrolizumab may result in a synergistic antitumor effect. Study design and methods: This is an open-label, pilot study (NCT03168438) of GSK3377794 in patients who are HLA-A*02:01, HLA-A*02:05, and/or HLA-A*02:06 positive and have NY-ESO-1/LAGE-1a positive relapsed/refractory MM. Twenty patients who have received at least three prior therapies containing at least one of the following drug classes as separate or combined lines of therapy (including ASCT): an immunomodulatory imide, proteasome inhibitor, alkylator, CD38 monoclonal antibody, and glucocorticoid, will be assigned to one of two arms: GSK3377794 alone as a single infusion (Arm 1, n=10) or GSK3377794 as a single infusion in combination with pembrolizumab 200 mg IV every 3 weeks (Arm 2, n=10). Enrollment of Arm 1 will be completed before enrolling subjects to Arm 2. Administration of pembrolizumab will start from Week 3 (or Week 6 if toxicities preclude Week 3 treatment). Patients will undergo leukapheresis to obtain cells for the manufacture of autologous NY-ESO-1-specific T cells. Each patient will then undergo lymphodepleting chemotherapy with fludarabine and cyclophosphamide followed by GSK3377794 infusion of 1−8x109 transduced T cells. Primary and secondary objectives are to assess safety and tolerability, and antitumor activity, respectively, of GSK3377794 treatment (with and without pembrolizumab). Patients will be monitored for adverse events and combination-related treatment-limiting toxicities; efficacy will be assessed using International Myeloma Working Group Response Criteria (Rajkumar et al, Blood 2011). In Arm 2, enrollment will be temporarily paused for a 3-week safety review period after the first 3 patients have received their first dose of pembrolizumab. Efficacy, safety, and biomarker assessments will be conducted at each visit. Patients will complete the treatment phase upon progression of disease or 108 weeks after GSK3377794 infusion. After completion of the treatment phase, patients will transfer to the long-term follow-up study (NCT03391778) to continue safety and survival monitoring for up to 15 years. As of January 27, 2019, 50 patients have undergone screening for HLA status and NY-ESO-1/LAGE-1a antigen expression. Among 50 patients screened for HLA, 25 (50%) tested positive for HLA-A*02:01, 05, and/or 06. Of these patients, bone marrow samples from 12/21 (57%) tested positive for NY-ESO-1, LAGE-1a, or both, illustrating high expression of this antigen in MM. To date, 3 patients have been treated with GSK3377794, demonstrating feasibility of identifying and treating HLA/antigen-positive patients with relapsed/refractory MM. Further work is underway towards introducing flexibility in screening procedures in order to permit wider screening of patients and to minimize time between screening and leukapheresis and for cell manufacturing, which will enhance patient eligibility. Acknowledgment: Medical writing support by O Conn PhD of Fishawack Indicia Ltd, funded by GSK. This study (NCT03168438) is funded by GSK. Disclosures Nishihori: Karyopharm: Research Funding; Novartis: Research Funding. Kaufman:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; AbbVie: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Winship Cancer Institute of Emory University: Employment; TG Therapeutics: Consultancy; Bristol-Myers Squibb: Consultancy; Incyte: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Blouch:GSK: Employment, Equity Ownership. Pandit:GSK: Employment, Equity Ownership. Butler:GSK: Employment, Equity Ownership. Jain:GSK: Employment. Wu:GSK: Employment, Equity Ownership. DeYoung:GSK: Employment, Equity Ownership. Hasan:GSK: Employment, Equity Ownership; Atara Biotherapeutics: Patents & Royalties; Merck: Equity Ownership