High prevalence of PCV2d in Hunan province, China: a retrospective analysis of samples collected from 2006 to 2016.

Porcine circovirus 2 (PCV2) has been widely prevailing in China since the first report in 2001, causing huge economic losses to the pig industry. In the present study, 674 samples were collected from 2006 to 2016 in Hunan province, and 62% were positive for PCV2. An increase was observed from 2006 to 2011 (72.1%-89.1%), and a decrease was observed from 2012 to 2016 (78.9%-36.8%). The prevalence of genotype PCV2a, PCV2b, and PCV2d was 0, 44.7% and 67%, respectively. During 2006-2007, PCV2b was the main genotype circulating in Hunan, while, in 2008, PCV2d became the predominant one. Coinfection with PCV2b and PCV2d was observed frequently, and the positive rates of coinfection ranged from 6.3% to 18.9% during 2006-2016. The complete genome was sequenced for 54 positive samples, and four were identified as PCV2b-1, 22 as PCV2b-2, four as PCV2d-1 and 24 as PCV2d-2, based on phylogenetic analysis of the complete genome and ORF2 region. Recombination analysis using the complete genome sequences of these isolates revealed a high recombination rate of 27.7% (17/54), and showed that recombination occurred mainly in the ORF1 region. This shows that the prevalence of PCV2 has clearly decreased in recent years and that PCV2d has become a predominant genotype since 2008. In addition, frequent recombination events were observed in the PCV2 isolates from Hunan, China.

Molecular characterization of Porcine sapelovirus in Hunan, China.

Porcine sapeloviruses (PSVs) are widely distributed in pig populations; however, little information on their evolutionary history and the mechanisms driving their divergence is available. Therefore, in the present study, 241 fecal samples and 91 intestinal contents collected from pigs at 26 farms in Hunan, China, were tested for the presence of PSVs. The overall PSV positivity rate was 46.39 %, with a particularly high infection rate detected in nursery and fattening pigs. A total of 29 PSV strains (PSV-HuNs) were isolated, with these showing high genetic diversity based on phylogenetic and pairwise distance analyses of the capsid-protein gene sequences. Incongruence between phylognetic trees of the capsid-protein and 3CD regions indicated frequent recombination within the PSV-HuNs, and a putative recombinant hotspot near the 3' end of the P1 region was identified. Our results suggested that recombination played an important role in driving PSV genetic diversity and evolution.

First isolation and genetic characteristics of porcine sapeloviruses in Hunan, China.

Outbreaks of diarrhea in piglets cause serious economic consequences in China. Diarrhetic fecal samples from 20 Hunan farm piglets were tested and found to be positive for porcine epidemic diarrhea virus (PEDV) by RT-PCR, although incubation with porcine kidney (PK-15) cells failed to produce infectious PEDV. Four porcine sapelovirus (PSV) strains (designated as PSV-HuNs) were isolated from four of the samples. Genomic sequence analysis revealed open reading frames encoding polyproteins of 2,331 (HuN1, 2 and 3) and 2,332 (HuN4) amino acids. Homology comparisons of the VP1 gene of the four Hunan strains with previously reported PSV strains revealed nucleotide sequence identities ranging from 74.2 to 98.6%, and deduced amino acid sequence identities from 79.5 to 98%. Phylogenetic analyses based on full-length and partial VP1 gene sequences showed that 3 of the PSV-HuN strains (HuN2, 3 and 4) clustered within a clade distinct from HuN1 as well as from all PSVs previously isolated in China, thereby showing that genetic diversity exists within Chinese PSVs. In addition, recombination analysis among PSVs indicates that a recombinant (HuN2 strain) exist in China.

Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed.

Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved significant clinical success. However, only a portion of patients benefit from T cell-based immunotherapy. Macrophages, the most abundant type of innate immune cells in the body, play an important role in eliminating tumor cells and infectious microbes. The phagocytic check point protein CD47 inhibits the phagocytic activity of macrophages through binding to SIRPα expressed on macrophages. Blockade of the interaction between CD47 and SIRPα could restore phagocytic activity and eliminate tumor cells in vitro and in vivo. In this manuscript, we review the mechanism of action and development status of agents (antibodies targeting CD47 and SIRPα, SIRPα-Fc fusion proteins, and bi-specific antibodies) that block CD47/SIRPα interaction in preclinical studies and in the clinical setting. In addition, small molecules, mRNA, and CAR-T/M that target the CD47/SIRPα axis are also reviewed in this article.

Penpulimab, an Fc-Engineered IgG1 Anti-PD-1 Antibody, With Improved Efficacy and Low Incidence of Immune-Related Adverse Events.

Background: IgG4 anbibodies are deficient in stability and may contribute to tumor-associated escape from immune surveillance. We developed an IgG1 backbone anti-programmed cell death protein-1 (PD-1) antibody, penpulimab, which is designed to remove crystallizable fragment (Fc) gamma receptor (FcγR) binding that mediates antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and proinflammatory cytokine release. Methods: Aggregation of different anti-PD-1 antibodies was tested by size exclusion chromatography, and melting temperature midpoint (Tm) and aggregation temperature onset (Tagg) were also determined. The affinity constants of penpulimab for PD-1 and human FcγRs were measured by surface plasmon resonance and biolayer interferometry. ADCC and ADCP were determined in cellular assays and antibody-dependent cytokine release (ADCR) from human macrophages was detected by ELISA. Binding kinetics of penpulimab to human PD-1 was determined by Biacore, and epitope/paratope mapping of PD-1/penpulimab was investigated using x-ray crystallography. Additionally, patients from six ongoing trials were included for analysis of immune-related adverse events (irAEs). Results: Penpulimab demonstrated better stability and a lower level of host-cell protein residue compared with IgG4 backbone anti-PD-1 antibodies. As expected, penpulimab exhibited no apparent binding to FcγRIa, FcγRIIa_H131, FcγRIIIa_V158 and FcγRIIIa_F158, elicited no apparent ADCC and ADCP activities, and induced no remarkable IL-6 and IL-8 release by activated macrophages in vitro. Penpulimab was shown in the co-crystal study to bind to human PD-1 N-glycosylation site at N58 and had a slower off-rate from PD-1 versus nivolumab or pembrolizumab. Four hundred sixty-five patients were analyzed for irAEs. Fifteen (3.2%) patients had grade 3 or above irAEs. No death from irAEs was reported. Conclusions: IgG1 backbone anti-PD1 antibody penpulimab has a good stability and reduced host cell protein residue, as well as potent binding to the antigen. Fc engineering has eliminated Fc-mediated effector functions of penpulimab including ADCC, ADCP and reduced ADCR, which may contribute to its more favorable safety profile. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: AK105-101: NCT03352531, AK105-201: NCT03722147, AK105-301: NCT03866980, AK105-202:NCT03866967, AK105-203: NCT04172571, AK105-204: NCT04172506.

Ligufalimab, a novel anti-CD47 antibody with no hemagglutination demonstrates both monotherapy and combo antitumor activity.

BACKGROUND: CD47 is a widely expressed transmembrane glycoprotein that delivers an antiphagocytic signal on macrophages through its interaction with SIRPα. CD47 is highly expressed in cancer cells and its overexpression is correlated with poor prognosis. CD47 blocking antibodies are actively being developed worldwide for cancer therapy, and the most challenging concern is associated with hematotoxicity. Ligufalimab (AK117) is a novel humanized IgG4 anti-CD47 antibody without hemagglutination effect. Blockade of CD47-SIRPα pathway by AK117 leads to a promising therapeutic strategy for cancer treatment with unique safety features. METHODS: AK117 was discovered through a screening hierarchy excluding hemagglutination. AK117 was characterized by detecting CD47-SIRPα blocking potential. Its effect on human red blood cells was examined and the mechanism of its binding with erythrocytes was studied. The abilities of AK117 and its combination with various opsonizing antibodies to promote macrophage-dependent phagocytosis of multiple human tumor cells were determined using fluorescence microscopy and flow cytometry. In vivo, the antitumor efficacy of AK117 monotherapy and combination with AK112 (an anti-PD-1/VEGF-A bispecific antibody) was assessed in a variety of xenograft models. Toxicologic studies were evaluated in non-human primates. RESULTS: AK117 bound to CD47 with high affinity and blocked the CD47-SIRPα interaction. AK117 did not induce hemagglutination and showed significantly lower degree of erythrophagocytosis compared with Hu5F9-G4, and this mechanism of hemagglutination resistance might be related to the binding conformation. AK117 enhanced macrophage-mediated phagocytosis in both hematologic cancer and solid tumor cell lines as a single agent or in combination with cetuximab and rituximab in vitro, respectively. The antitumor effects of AK117 as a single agent or in combination with AK112 were also encouraging in various xenograft models. In non-human primates, AK117 showed less hematotoxicity compared with Hu5F9-G4. CONCLUSIONS: AK117 eliminated hemagglutination and also enabled to maintain full effectiveness of CD47 blockade on tumor cells, which resulted in excellent antitumor efficacy and favorable safety profile of AK117. A series of clinical trials of AK117 as a therapeutic agent in combination with various agents such as AK112 are in progress for the treatment of multiple hematologic malignancies and solid tumors.

Construction of an HRP-streptavidin bound antigen and its application in an ELISA for porcine circovirus 2 antibodies.

A fusion protein SBP-Cap∆41, consisting of Cap∆41 (without 41 amino acids at the N-terminus) protein of porcine circovirus 2 (PCV2) and a streptavidin binding peptide (SBP), was constructed. This fusion protein binds to HRP-labeled streptavidin (HRP-SA) through high affinity between SBP and SA, forming an HRP-streptavidin bound antigen (Hsb-Ag) with both immunoreactivity and enzymatic activity, which can be used in a double-antigen sandwich ELISA for detection of PCV2 antibodies. Comparison of the characteristics of the HSb-Cap∆41 and chemical conjugates of the recombinant Cap∆41 protein showed that the HSb-Cap∆41 based double-antigen sandwich ELISA (HBDS-ELISA) had higher specificity and sensitivity. Use of the HBDS-ELISA detected PCV2-IgG in 9 injected pigs as early as 10 days p.i., 3 days earlier than both a double-antigen sandwich ELISA (DS-ELISA) based on a chemically conjugated antigen, and a commercial indirect ELISA kit.