Complement inhibitors for age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a common eye disease and leading cause of sight loss worldwide. Despite its high prevalence and increasing incidence as populations age, AMD remains incurable and there are no treatments for most patients. Mounting genetic and molecular evidence implicates complement system overactivity as a key driver of AMD development and progression. The last decade has seen the development of several novel therapeutics targeting complement in the eye for the treatment of AMD. This review update encompasses the results of the first randomised controlled trials in this field. OBJECTIVES: To assess the effects and safety of complement inhibitors in the prevention or treatment of AMD. SEARCH METHODS: We searched CENTRAL on the Cochrane Library, MEDLINE, Embase, LILACS, Web of Science, ISRCTN registry, ClinicalTrials.gov, and the WHO ICTRP to 29 June 2022 with no language restrictions. We also contacted companies running clinical trials for unpublished data. SELECTION CRITERIA: We included randomised controlled trials (RCTs) with parallel groups and comparator arms that studied complement inhibition for advanced AMD prevention/treatment. DATA COLLECTION AND ANALYSIS: Two authors independently assessed search results and resolved discrepancies through discussion. Outcome measures evaluated at one year included change in best-corrected visual acuity (BCVA), untransformed and square root-transformed geographic atrophy (GA) lesion size progression, development of macular neovascularisation (MNV) or exudative AMD, development of endophthalmitis, loss of ≥ 15 letters of BCVA, change in low luminance visual acuity, and change in quality of life. We assessed risk of bias and evidence certainty using Cochrane risk of bias and GRADE tools. MAIN RESULTS: Ten RCTs with 4052 participants and eyes with GA were included. Nine evaluated intravitreal (IVT) administrations against sham, and one investigated an intravenous agent against placebo. Seven studies excluded patients with prior MNV in the non-study eye, whereas the three pegcetacoplan studies did not. The risk of bias in the included studies was low overall. We also synthesised results of two intravitreal agents (lampalizumab, pegcetacoplan) at monthly and every-other-month (EOM) dosing intervals. Efficacy and safety of IVT lampalizumab versus sham for GA For 1932 participants in three studies, lampalizumab did not meaningfully change BCVA given monthly (+1.03 letters, 95% confidence interval (CI) -0.19 to 2.25) or EOM (+0.22 letters, 95% CI -1.00 to 1.44) (high-certainty evidence). For 1920 participants, lampalizumab did not meaningfully change GA lesion growth given monthly (+0.07 mm², 95% CI -0.09 to 0.23; moderate-certainty due to imprecision) or EOM (+0.07 mm², 95% CI -0.05 to 0.19; high-certainty). For 2000 participants, lampalizumab may have also increased MNV risk given monthly (RR 1.77, 95% CI 0.73 to 4.30) and EOM (RR 1.70, 95% CI 0.67 to 4.28), based on low-certainty evidence. The incidence of endophthalmitis in patients treated with monthly and EOM lampalizumab was 4 per 1000 (0 to 87) and 3 per 1000 (0 to 62), respectively, based on moderate-certainty evidence. Efficacy and safety of IVT pegcetacoplan versus sham for GA For 242 participants in one study, pegcetacoplan probably did not meaningfully change BCVA given monthly (+1.05 letters, 95% CI -2.71 to 4.81) or EOM (-1.42 letters, 95% CI -5.25 to 2.41), as supported by moderate-certainty evidence. In contrast, for 1208 participants across three studies, pegcetacoplan meaningfully reduced GA lesion growth when given monthly (-0.38 mm², 95% CI -0.57 to -0.19) and EOM (-0.29 mm², 95% CI -0.44 to -0.13), with high certainty. These reductions correspond to 19.2% and 14.8% versus sham, respectively. A post hoc analysis showed possibly greater benefits in 446 participants with extrafoveal GA given monthly (-0.67 mm², 95% CI -0.98 to -0.36) and EOM (-0.60 mm², 95% CI -0.91 to -0.30), representing 26.1% and 23.3% reductions, respectively. However, we did not have data on subfoveal GA growth to undertake a formal subgroup analysis. In 1502 participants, there is low-certainty evidence that pegcetacoplan may have increased MNV risk when given monthly (RR 4.47, 95% CI 0.41 to 48.98) or EOM (RR 2.29, 95% CI 0.46 to 11.35). The incidence of endophthalmitis in patients treated with monthly and EOM pegcetacoplan was 6 per 1000 (1 to 53) and 8 per 1000 (1 to 70) respectively, based on moderate-certainty evidence. Efficacy and safety of IVT avacincaptad pegol versus sham for GA In a study of 260 participants with extrafoveal or juxtafoveal GA, monthly avacincaptad pegol probably did not result in a clinically meaningful change in BCVA at 2 mg (+1.39 letters, 95% CI -5.89 to 8.67) or 4 mg (-0.28 letters, 95% CI -8.74 to 8.18), based on moderate-certainty evidence. Despite this, the drug was still found to have probably reduced GA lesion growth, with estimates of 30.5% reduction at 2 mg (-0.70 mm², 95% CI -1.99 to 0.59) and 25.6% reduction at 4 mg (-0.71 mm², 95% CI -1.92 to 0.51), based on moderate-certainty evidence. Avacincaptad pegol may have also increased the risk of developing MNV (RR 3.13, 95% CI 0.93 to 10.55), although this evidence is of low certainty. There were no cases of endophthalmitis reported in this study. AUTHORS' CONCLUSIONS: Despite confirmation of the negative findings of intravitreal lampalizumab across all endpoints, local complement inhibition with intravitreal pegcetacoplan meaningfully reduces GA lesion growth relative to sham at one year. Inhibition of complement C5 with intravitreal avacincaptad pegol is also an emerging therapy with probable benefits on anatomical endpoints in the extrafoveal or juxtafoveal GA population. However, there is currently no evidence that complement inhibition with any agent improves functional endpoints in advanced AMD; further results from the phase 3 studies of pegcetacoplan and avacincaptad pegol are eagerly awaited. Progression to MNV or exudative AMD is a possible emergent adverse event of complement inhibition, requiring careful consideration should these agents be used clinically. Intravitreal administration of complement inhibitors is probably associated with a small risk of endophthalmitis, which may be higher than that of other intravitreal therapies. Further research is likely to have an important impact on our confidence in the estimates of adverse effects and may change these. The optimal dosing regimens, treatment duration, and cost-effectiveness of such therapies are yet to be established.

Comparison of the use of internal limiting membrane flaps versus conventional ILM peeling on post-operative anatomical and visual outcomes in large macular holes.

BACKGROUND: Idiopathic full-thickness macular hole (iFTMH) closure rates following conventional vitrectomy, gas tamponade and internal limiting membrane (ILM) peeling decrease when the minimum linear diameter (MLD) ≥ 500 microns. ILM flap creation has been proposed to improve closure in larger holes. This study evaluated the anatomical and functional impact of ILM flap introduction to routine practice in iFTMH ≥500 microns. METHODS: Retrospective, interventional analysis of prospectively collected data of 191 eyes from consecutive surgeries for primary iFTMH ≥500 microns performed by two surgeons between June 2018 and June 2022, during which both surgeons replaced ILM peeling with ILM flap creation. Post-operative best-corrected visual acuity (BCVA) and anatomical closure were compared between Group 1 (ILM peel) and Group 2 (ILM flap) in an intention-to-treat analysis. RESULTS: Rates of iFTMH closure were greater in the ILM flap group (77/80; 96.3%) than the ILM peel group (94/110; 85.5%) (OR = 4.37, 95% CI = 1.23-15.55, p = 0.023). A non-significant increase in post-operative BCVA improvement was observed in the ILM flap group (p = 0.084). There was no statistically significant difference in final BCVA (p = 0.83). Multivariate logistic regression found only MLD (OR = 0.993, 95% CI = 0.989-0.997, p = 0.001) and ILM flap group (OR = 5.795, 95% CI = 1.313-25.570, p = 0.020) predicted primary closure. CONCLUSION: ILM flap creation improves closure rates in larger holes and should be considered routinely in iFTMH ≥500 microns. Whether ILM flaps affect post-operative visual function remains uncertain.

Differential proteomic analysis of bovine conceptus fluid proteins in pregnancies generated by assisted reproductive technologies.

Proteomic analysis of bovine conceptus fluid proteins during early pregnancy has the potential to expose protein species indicative of both the overall health of the fetal-maternal environment and fetal developmental status. In this study, we examined the differential abundance of bovine conceptus fluid proteins (5-50 kDa fraction) from naturally conceived, in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT)-derived pregnancies at days 45 and 90 of gestation. In day 45 allantoic fluid (AllF) samples, an atypical cluster of low molecular weight ( approximately 14-16 kDa), low pI (between 3.0 and 4.5 pH units) protein species was increased in three of four IVF samples (30-100-fold increase in protein spot volumes compared to normal). These proteins were identified as paralogs of the bovine cathelicidin antimicrobial protein (CAMP) by MALDI-TOF MS peptide mass fingerprint and MALDI-TOF MS/MS peptide sequence analysis. Peptidoglycan recognition protein and serine (or cysteine) proteinase inhibitor clade B1, were also significantly increased in the corresponding IVF samples. In two of four SCNT AllF samples, a 2-10-fold increase in CAMP protein spot volumes were detected. No aberrant abundance levels of individual protein species were observed in amniotic fluid samples, or in day 90 IVF AllF samples. Identification of unique protein species present in the normal bovine AllF proteome at day 45 is also reported.

Conceptus-related measurements during the first trimester of bovine pregnancy.

In order to determine the variability inherent in conceptus-related measurements in first trimester bovine pregnancies, conceptus and fetometric parameters from beef cattle pregnancies (n=103) estimated to be between Days 36 and 103 of gestation were examined. During this period, the protein concentration of amniotic fluid ranged between 0.181 and 0.501mg/mL. The amniotic fluid volume gradually increased from <1mL at Day 36 to 950mL at Day 103 (R(2)=0.9275) and amniotic compartment dimensions (length, R(2)=0.9713; width, R(2)=0.9802) increased predictably with fetal growth. Conversely, allantoic fluid protein concentration and volume correlated weakly with fetal age. A significant linear correlation existed between fetal crown rump length (CRL) and crown nose length (R(2)=0.9899) confirming that either measurement can be employed in the ultrasonographic estimation of fetal age. The amniotic compartment and fetometric data presented here have both research and clinical value, particularly in relation to fetal development evaluation and pregnancy viability diagnosis.

Proteomic analysis of bovine conceptus fluids during early pregnancy.

A proteomic analysis of bovine amniotic and allantoic fluids collected around Day 45 of gestation was performed using gel-based and LC-based MS workflows. A depletion/enrichment protocol using ultrafiltration under denaturing and reducing conditions produced an enriched fraction containing protein species predominantly between 5 and 50 kDa molecular weight. The analyses of conceptus fluid proteins were performed using two strategies; first, 2-DE coupled with MALDI-TOF-MS/MS and LC-ESI-MS/MS analysis of individual protein spots and second, a global protein snapshot of the enriched 5-50 kDa protein fraction by LC-ESI-MS/MS and LC-MALDI-TOF-MS/MS. Allocation of bovine specific protein identities was achieved by searching the Interactive Bovine In Silico SNP (IBISS) and NCBInr protein sequence databases resulting in the confident PMF identification and MS/MS confirmation of >200 2-DE generated allantoic fluids protein spots (74 individual protein species identified) and the MS/MS peptide identification of 105 LC-ESI-MS/MS generated protein identities. In total, the identity of 139 individual protein species from allantoic fluids was confirmed with peptide sequence probability MOWSE scores at the p<0.05 level or better. The comparison of bovine Day 45 amniotic and allantoic fluids protein profiles revealed differences between these two conceptus fluids in early pregnancy.

TaNAC69 from the NAC superfamily of transcription factors is up-regulated by abiotic stresses in wheat and recognises two consensus DNA-binding sequences.

NAC proteins are one of the largest families of plant transcription factors and have recently been implicated in diverse physiological processes. To elucidate their role in gene regulation, we determined the DNA-binding specificity of a drought- and cold-inducible NAC protein, TaNAC69 from wheat, and analysed its homologues from other species. Two consensus DNA-binding sequences (spanning 23-24 bp) of TaNAC69 were identified through binding site selection and both consisted of two half sites. Comprehensive data on the DNA-binding specificity of TaNAC69 were generated through extensive base substitution mutagenesis. TaNAC69 and its homologue in Arabidopsis, NAP, sharing 75% sequence identity in the NAC domain, exhibited similar DNA-binding specificity. TaNAC69 was able to homodimerise through its NAC domain. The NAC domain consists of five conserved subdomains. Subdomain mutation showed that a loss or reduction in TaNAC69 dimerisation capacity was accompanied with abolition or decrease in its DNA-binding activity. These data suggest that all subdomains are necessary to maintain a functional NAC domain structure required for interaction with DNA and dimerisation.

Identification of novel protective antigens from Anaplasma marginale.

A successful recombinant vaccine against Anaplasma marginale remains elusive, despite intensive study of the protective, though variable major surface antigens. As an alternative approach to the discovery of additional antigens, crude parasite material was subjected to conventional protein fractionation, coupled with vaccination and parasite challenge trials, without making assumptions as to the nature or location of these antigens. This has lead to the identification of four novel antigens that, in microgram amounts, have significant protective effects in vaccination trials. The antigens have molecular weights in the range 17-43 kDa and isoelectric points above 7.8. Limited N-terminal and internal sequencing of these antigens has established that they are parasite proteins previously unreported in the literature, although one of the four is identifiable with a recently reported open reading frame of unknown function.

A novel antigen from Anaplasma marginale: characterization, expression and preliminary evaluation of the recombinant protein.

Through a process of protein fractionation and vaccination we previously identified four native antigens that confer a degree of protection against challenge with Anaplasma marginale. One of these, Ana 29 has been successfully cloned and sequenced using degenerate primers designed to N-terminal and internal peptide sequences. The full-length gene codes for a protein with a theoretical molecular weight of 27 kDa and pI 8.6. The sequence is highly conserved, showing 99% identity between two Australian and an American isolate of A. marginale. The gene sequences from these isolates also share 99% identity with the strain of Anaplasma centrale used in the commercial Australian vaccine. Protein prediction algorithms suggest the native protein is an integral membrane protein. This protein has been over-expressed and purified from Escherichia coli and used in vaccination trials in cattle using two adjuvants. The initial results from the trial show a significant level of protection was obtained with one adjuvant; in comparison, the second adjuvant slightly aggravated the disease. Preliminary data suggests a good correlation between the induction of an IgG2 response and protection.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria.

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4(+) T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-alpha, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.